Comparative ligand mapping from MHC positive cells

ABSTRACT

The present invention relates generally to a methodology for the isolation, purification and identification of peptide ligands presented by MHC positive cells. In particular, the methodology of the present invention relates to the isolation, purification and identification of these peptide ligands from soluble class I and class II MHC molecules which may be uninfected, infected, or tumorgenic. The methodology of the present invention broadly allows for these peptide ligands and their comcomittant source proteins thereof to be identified and used as markers for infected versus uninfected cells and/or tumorgenic versus nontumorgenic cells with said identification being useful for marking or targeting a cell for therapeutic treatment or priming the immune response against infected cells.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates generally to a methodology for theisolation, purification and identification of peptide ligands presentedby MHC positive cells. In particular, the methodology of the presentinvention relates to the isolation, purification and identification ofthese peptide ligands from soluble class I and class II MHC moleculeswhich may be uninfected, infected, or tumorigenic. The methodology ofthe present invention broadly allows for these peptide ligands and theirconcomitant source proteins thereof to be identified and used as markersfor infected versus uninfected cells and/or tumorigenic versusnontumorigenic cells with said identification being useful for markingor targeting a cell for therapeutic treatment or priming the immuneresponse against infected cells.

[0003] 2. Description of the Background Art

[0004] Class I major histocompatibility complex (MHC) molecules,designated HLA class I in humans, bind and display peptide antigenligands upon the cell surface. The peptide antigen ligands presented bythe class I MHC molecule are derived from either normal endogenousproteins (“self”) or foreign proteins (“nonself”) introduced into thecell. Nonself proteins may be products of malignant transformation orintracellular pathogens such as viruses. In this manner, class I MHCmolecules convey information regarding the internal fitness of a cell toimmune effector cells including but not limited to, CD8⁺ cytotoxic Tlymphocytes (CTLs), which are activated upon interaction with “nonself”peptides, thereby lysing or killing the cell presenting such “nonself”peptides.

[0005] Class II MHC molecules, designated HLA class II in humans, alsobind and display peptide antigen ligands upon the cell surface. Unlikeclass I MHC molecules which are expressed on virtually all nucleatedcells, class II MHC molecules are normally confined to specializedcells, such as B lymphocytes, macrophages, dendritic cells, and otherantigen presenting cells which take up foreign antigens from theextracellular fluid via an endocytic pathway. The peptides they bind andpresent are derived from extracellular foreign antigens, such asproducts of bacteria that multiply outside of cells, wherein suchproducts include protein toxins secreted by the bacteria that oftentimes have deleterious and even lethal effects on the host (e.g. human).In this manner, class II molecules convey information regarding thefitness of the extracellular space in the vicinity of the celldisplaying the class II molecule to immune effector cells, including butnot limited to, CD4⁺ helper T cells, thereby helping to eliminate suchpathogens the examination of such pathogens is accomplished by bothhelping B cells make antibodies against microbes, as well as toxinsproduced by such microbes, and by activating macrophages to destroyingested microbes.

[0006] Class I and class II HLA molecules exhibit extensive polymorphismgenerated by systematic recombinatorial and point mutation events; assuch, hundreds of different HLA types exist throughout the world'spopulation, resulting in a large immunological diversity. Such extensiveHLA diversity throughout the population results in tissue or organtransplant rejection between individuals as well as differingsusceptibilities and/or resistances to infectious diseases. HLAmolecules also contribute significantly to autoimmunity and cancer.Because HLA molecules mediate most, if not all, adaptive immuneresponses, large quantities of pure isolated HLA proteins are requiredin order to effectively study transplantation, autoimmunity disorders,and for vaccine development.

[0007] There are several applications in which purified, individualclass I and class II MHC proteins are highly useful. Such applicationsinclude using MHC-peptide multimers as immunodiagnostic reagents fordisease resistance/autoimmunity; assessing the binding of potentiallytherapeutic peptides; elution of peptides from MHC molecules to identifyvaccine candidates; screening transplant patients for preformed MHCspecific antibodies; and removal of anti-HLA antibodies from a patient.Since every individual has differing MHC molecules, the testing ofnumerous individual MHC molecules is a prerequisite for understandingthe differences in disease susceptibility between individuals.Therefore, purified MHC molecules representative of the hundreds ofdifferent HLA types existing throughout the world's population arehighly desirable for unraveling disease susceptibilities andresistances, as well as for designing therapeutics such as vaccines.

[0008] Class I HLA molecules alert the immune response to disorderswithin host cells. Peptides, which are derived from viral- andtumor-specific proteins within the cell, are loaded into the class Imolecule's antigen binding groove in the endoplasmic reticulum of thecell and subsequently carried to the cell surface. Once the class I HLAmolecule and its loaded peptide ligand are on the cell surface, theclass I molecule and its peptide ligand are accessible to cytotoxic Tlymphocytes (CTL). CTL survey the peptides presented by the class Imolecule and destroy those cells harboring ligands derived frominfectious or neoplastic agents within that cell.

[0009] While specific CTL targets have been identified, little is knownabout the breadth and nature of ligands presented on the surface of adiseased cell. From a basic science perspective, many outstandingquestions have permeated through the art regarding peptide exhibition.For instance, it has been demonstrated that a virus can preferentiallyblock expression of HLA class I molecules from a given locus whileleaving expression at other loci intact. Similarly, there are numerousreports of cancerous cells that fail to express class I HLA atparticular loci. However, there are no data describing how (or if) thethree classical HLA class I loci differ in the immunoregulatory ligandsthey bind. It is therefore unclear how class I molecules from thedifferent loci vary in their interaction with viral- and tumor-derivedligands and the number of peptides each will present.

[0010] Discerning virus- and tumor-specific ligands for CTL recognitionis an important component of vaccine design. Ligands unique totumorigenic or infected cells can be tested and incorporated intovaccines designed to evoke a protective CTL response. Severalmethodologies are currently employed to identify potentially protectivepeptide ligands. One approach uses T cell lines or clones to screen forbiologically active ligands among chromatographic fractions of elutedpeptides. (Cox et al., Science, vol 264, 1994, pages 716-719, which isexpressly incorporated herein by reference in its entirety) Thisapproach has been employed to identify peptides ligands specific tocancerous cells. A second technique utilizes predictive algorithms toidentify peptides capable of binding to a particular class I moleculebased upon previously determined motif and/or individual ligandsequences. (De Groot et al., Emerging Infectious Diseases, (7) 4, 2001,which is expressly incorporated herein by reference in its entirety)Peptides having high predicted probability of binding from a pathogen ofinterest can then be synthesized and tested for T cell reactivity inprecursor, tetramer or ELISpot assays.

[0011] However, there has been no readily available source of individualHLA molecules. The quantities of HLA protein available have been smalland typically consist of a mixture of different HLA molecules.Production of HLA molecules traditionally involves growth and lysis ofcells expressing multiple HLA molecules. Ninety percent of thepopulation is heterozygous at each of the HLA loci; codominantexpression results in multiple HLA proteins expressed at each HLA locus.To purify native class I or class II molecules from mammalian cellsrequires time-consuming and cumbersome purification methods, and sinceeach cell typically expresses multiple surface-bound HLA class I orclass II molecules, HLA purification results in a mixture of manydifferent HLA class I or class II molecules. When performing experimentsusing such a mixture of HLA molecules or performing experiments using acell having multiple surface-bound HLA molecules, interpretation ofresults cannot directly distinguish between the different HLA molecules,and one cannot be certain that any particular HLA molecule isresponsible for a given result. Therefore, a need existed in the art fora method of producing substantial quantities of individual HLA class Ior class II molecules so that they can be readily purified and isolatedindependent of other HLA class I or class II molecules. Such individualHLA molecules, when provided in sufficient quantity and purity, wouldprovide a powerful tool for studying and measuring immune responses.

[0012] Therefore, there exists a need in the art for improved methods ofepitope discovery and comparative ligand mapping for class I and classII MHC molecules, including methods of distinguishing an infected/tumorcell from an uninfected/non-tumor cell. The present invention solvesthis need by coupling the production of soluble HLA molecules with anepitope isolation, discovery, and direct comparison methodology.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1. Overview of 2 stage PCR strategy to amplify a truncatedversion of the human class I MHC.

[0014]FIG. 2. Edman sequence analysis of soluble B*1501, B*1501-HIS andB*1501-FLAG. Residue intensity was categorized as either dominant(3.5-fold or more picomolar increase over previous round) or strong (2.5to 3.5-fold increase over prior round).

[0015]FIG. 3. Representative MS ion maps from soluble B*1501, B*1501-HISand B*1501-FLAG illustrating ion overlap between the molecules. Pooled,acid-eluted peptides were fractionated by RP-HPLC, and the individualfractions were MS scanned.

[0016]FIG. 4. Fragmentation pattern generated by MS/MS on an ionselected from fraction 11 of B*1501, B*1501-HIS and B*1501-FLAG peptidesillustrating a sequence-level overlap between the three molecules.

[0017]FIG. 5. Flow chart of the epitope discovery of C-terminal-taggedsHLA molecules. Class I positive transfectants are infected with apathogen of choice and sHLA preferentially purified utilizing the tag.Subtractive comparison of MS ion maps yields ions present only ininfected cell, which are then MS/MS sequenced to derive class Iepitopes.

[0018]FIG. 6. MS ion map from soluble B*0702 SupT1 cells uninfected andinfected with HIV MN-1. Pooled, acid-eluted peptides were fractionatedby RP-HPLC, and fraction #30 was MS scanned.

[0019]FIG. 7. MS ion map similar to FIG. 6 but zoomed in on the areafrom 482-488 amu to more clearly identify all ions in the immediatearea.

[0020]FIG. 8. Fragmentation pattern generated by tandem massspectrometry of the peptide ion 484.72 isolated from infected solubleB*0702 SupT1 cells.

[0021]FIG. 9. Results of a PubMed BLAST search with the sequenceGPRTAALGLL identified in FIG. 8.

[0022]FIG. 10. Summary of Results of Entrez-PubMed search for the word“reticulocalbin”.

[0023]FIG. 11. Results of a peptide-binding algorithm performed usingParker's Prediction using the entire source protein, reticulocalbin,which generates a list of peptides which are bound by the B*0702 HLAallele.

[0024]FIG. 12. Results of a peptide-binding algorithm performed usingRammensee's SYPEITHI Prediction using the entire source protein,reticulocalbin, which generates a list of peptides which are bound bythe B*0702 HLA allele.

[0025]FIG. 13. Results of a predicted proteasomal cleavage of thecomplete reticulocalbin protein using the cleavage predictor PaProC.

[0026]FIG. 14. Results of a predicted proteasomal cleavage of thecomplete reticulocalbin protein using the cleavage predictor NetChop2.0.

[0027]FIG. 15. Several high affinity peptides deriving fromreticulocalbin were identified as peptides predicted to be presented byHLA-A*0201 and A*0101.

[0028]FIG. 16. MS ion maps from soluble B*0702 uninfected SupT1 cells offractions 29 and 31 to determine that ion 484.72 was not present.

[0029]FIG. 17. Fragmentation patterns of soluble B*0702 uninfected SupT1cells fraction 30 ion 484.72 under identical MS collision conditions toillustrate the absence of the reticulocalbin peptide in the uninfectedcells.

[0030]FIG. 18. Comparison of the MS/MS fragmentation patterns ofsynthetic peptide GPRTAALGLL and peptide ion 484.72 isolated frominfected soluble B*0702 SupT1 cells.

DETAILED DESCRIPTION OF THE INVENTION

[0031] Before explaining at least one embodiment of the invention indetail by way of exemplary drawings, experimentation, results, andlaboratory procedures, it is to be understood that the invention is notlimited in its application to the details of construction and thearrangement of the components set forth in the following description orillustrated in the drawings, experimentation and/or results. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways. As such, the language used herein isintended to be given the broadest possible scope and meaning; and theembodiments are meant to be exemplary—not exhaustive. Also, it is to beunderstood that the phraseology and terminology employed herein is forthe purpose of description and should not be regarded as limiting.

[0032] The present invention generally relates to a method of epitopediscovery and comparative ligand mapping as well as methods ofdistinguishing infected/tumor cells from uninfected/non-tumor cells. Thepresent method broadly includes the following steps: (1) providing acell line containing a construct that encodes an individual solubleclass I or class II MHC molecule (wherein the cell line is capable ofnaturally processing self or nonself proteins into peptide ligandscapable of being loaded into the antigen binding grooves of the class Ior class II MHC molecules); (2) culturing the cell line under conditionswhich allow for expression of the individual soluble class I or class IIMHC molecule from the construct, with such conditions also allowing forthe endogenous loading of a peptide ligand (from the self or non-selfprocessed protein) into the antigen binding groove of each individualsoluble class I or class II MHC molecule prior to secretion of thesoluble class I or class II MHC molecules having the peptide ligandsbound thereto; and (4) separating the peptide ligands from theindividual soluble class I or class II MHC molecules.

[0033] The methods of the present invention may, in one embodiment,utilize a method of producing MHC molecules (from genomic DNA or cDNA)that are secreted from mammalian cells in a bioreactor unit. Substantialquantities of individual MHC molecules are obtained by modifying class Ior class II MHC molecules so that they are capable of being secreted,isolated, and purified. Secretion of soluble MHC molecules overcomes thedisadvantages and defects of the prior art in relation to the quantityand purity of MHC molecules produced. Problems of quantity are overcomebecause the cells producing the MHC do not need to be detergent lysed orkilled in order to obtain the MHC molecule. In this way the cellsproducing secreted MHC remain alive and therefore continue to produceMHC. Problems of purity are overcome because the only MHC moleculesecreted from the cell is the one that has specifically been constructedto be secreted. Thus, transfection of vectors encoding such secreted MHCmolecules into cells which may express endogenous, surface bound MHCprovides a method of obtaining a highly concentrated form of thetransfected MHC molecule as it is secreted from the cells. Greaterpurity is assured by transfecting the secreted MHC molecule into MHCdeficient cell lines.

[0034] Production of the MHC molecules in a hollow fiber bioreactor unitallows cells to be cultured at a density substantially greater thanconventional liquid phase tissue culture permits. Dense culturing ofcells secreting MHC molecules further amplifies the ability tocontinuously harvest the transfected MHC molecules. Dense bioreactorcultures of MHC secreting cell lines allow for high concentrations ofindividual MHC proteins to be obtained. Highly concentrated individualMHC proteins provide an advantage in that most downstream proteinpurification strategies perform better as the concentration of theprotein to be purified increases. Thus, the culturing of MHC secretingcells in bioreactors allows for a continuous production of individualMHC proteins in a concentrated form.

[0035] The method of producing MHC molecules utilized in the presentinvention begins by obtaining genomic or complementary DNA which encodesthe desired MHC class I or class II molecule. Alleles at the locus whichencode the desired MHC molecule are PCR amplified in a locus specificmanner. These locus specific PCR products may include the entire codingregion of the MHC molecule or a portion thereof. In one embodiment anested or hemi-nested PCR is applied to produce a truncated form of theclass I or class II gene so that it will be secreted rather thananchored to the cell surface. In another embodiment the PCR willdirectly truncate the MHC molecule.

[0036] Locus specific PCR products are cloned into a mammalianexpression vector and screened with a variety of methods to identify aclone encoding the desired MHC molecule. The cloned MHC molecules areDNA sequenced to insure fidelity of the PCR. Faithful truncated clonesof the desired MHC molecule are then transfected into a mammalian cellline. When such cell line is transfected with a vector encoding arecombinant class I molecule, such cell line may either lack endogenousclass I MHC molecule expression or express endogenous class I MHCmolecules. One of ordinary skill of the art would note the importance,given the present invention, that cells expressing endogenous class IMHC molecules may spontaneously release MHC into solution upon naturalcell death. In cases where this small amount of spontaneously releasedMHC is a concern, the transfected class I MHC molecule can be “tagged”such that it can be specifically purified away from spontaneouslyreleased endogenous class I molecules in cells that express class Imolecules. For example, a DNA fragment encoding a HIS tail may beattached to the protein by the PCR reaction or may be encoded by thevector into which the PCR fragment is cloned, and such HIS tail,therefore, further aids in the purification of the class I MHC moleculesaway from endogenous class I molecules. Tags beside a histidine tailhave also been demonstrated to work, and one of ordinary skill in theart of tagging proteins for downstream purification would appreciate andknow how to tag a MHC molecule in such a manner so as to increase theease by which the MHC molecule may be purified.

[0037] Cloned genomic DNA fragments contain both exons and introns aswell as other non-translated regions at the 5′ and 3′ termini of thegene. Following transfection into a cell line which transcribes thegenomic DNA (gDNA) into RNA, cloned genomic DNA results in a proteinproduct thereby removing introns and splicing the RNA to form messengerRNA (mRNA), which is then translated into an MHC protein. Transfectionof MHC molecules encoded by gDNA therefore facilitates reisolation ofthe gDNA, mRNA/cDNA, and protein. Production of MHC molecules innon-mammalian cell lines such as insect and bacterial cells requirescDNA clones, as these lower cell types do not have the ability to spliceintrons out of RNA transcribed from a gDNA clone. In these instances themammalian gDNA transfectants of the present invention provide a valuablesource of RNA which can be reverse transcribed to form MHC cDNA. ThecDNA can then be cloned, transferred into cells, and then translatedinto protein. In addition to producing secreted MHC, such gDNAtransfectants therefore provide a ready source of mRNA, and thereforecDNA clones, which can then be transfected into non-mammalian cells forproduction of MHC. Thus, the present invention which starts with MHCgenomic DNA clones allows for the production of MHC in cells fromvarious species.

[0038] A key advantage of starting from gDNA is that viable cellscontaining the MHC molecule of interest are not needed. Since allindividuals in the population have a different MHC repertoire, one wouldneed to search more than 500,000 individuals to find someone with thesame MHC complement as a desired individual—such a practical example ofthis principle is observed when trying to find a donor to match arecipient for bone marrow transplantation. Thus, if it is desired toproduce a particular MHC molecule for use in an experiment ordiagnostic, a person or cell expressing the MHC allele of interest wouldfirst need to be identified. Alternatively, in the method of the presentinvention, only a saliva sample, a hair root, an old freezer sample, orless than a milliliter (0.2 ml) of blood would be required to isolatethe gDNA. Then, starting from gDNA, the MHC molecule of interest couldbe obtained via a gDNA clone as described herein, and followingtransfection of such clone into mammalian cells, the desired proteincould be produced directly in mammalian cells or from cDNA in severalspecies of cells using the methods of the present invention describedherein.

[0039] Current experiments to obtain an MHC allele for proteinexpression typically start from mRNA, which requires a fresh sample ofmammalian cells that express the MHC molecule of interest. Working fromgDNA does not require gene expression or a fresh biological sample. Itis also important to note that RNA is inherently unstable and is not aseasily obtained as is gDNA. Therefore, if production of a particular MHCmolecule starting from a cDNA clone is desired, a person or cell linethat is expressing the allele of interest must traditionally first beidentified in order to obtain RNA. Then a fresh sample of blood or cellsmust be obtained; experiments using the methodology of the presentinvention show that ≧5 milliliters of blood that is less than 3 days oldis required to obtain sufficient RNA for MHC cDNA synthesis. Thus, bystarting with gDNA, the breadth of MHC molecules that can be readilyproduced is expanded. This is a key factor in a system as polymorphic asthe MHC system; hundreds of MHC molecules exist, and not all MHCmolecules are readily available. This is especially true of MHCmolecules unique to isolated populations or of MHC molecules unique toethnic minorities. Starting class I or class II MHC molecule expressionfrom the point of genomic DNA simplifies the isolation of the gene ofinterest and insures a more equitable means of producing MHC moleculesfor study; otherwise, one would be left to determine whose MHC moleculesare chosen and not chosen for study, as well as to determine whichethnic population from which fresh samples cannot be obtained andtherefore should not have their MHC molecules included in a diagnosticassay.

[0040] While cDNA may be substituted for genomic DNA as the startingmaterial, production of cDNA for each of the desired HLA class I typeswill require hundreds of different, HLA typed, viable cell lines, eachexpressing a different HLA class I type. Alternatively, fresh samplesare required from individuals with the various desired MHC types. Theuse of genomic DNA as the starting material allows for the production ofclones for many HLA molecules from a single genomic DNA sequence, as theamplification process can be manipulated to mimic recombinatorial andgene conversion events. Several mutagenesis strategies exist whereby agiven class I gDNA clone could be modified at either the level of gDNAor at the cDNA resulting from this gDNA clone. The process of producingMHC molecules utilized in the present invention does not require viablecells, and therefore the degradation which plagues RNA is not a problem.

[0041] The soluble class I MHC proteins produced by the method describedherein is utilized in the methods of epitope discovery and comparativeligand mapping of the present invention. The methods of epitopediscovery and comparative ligand mapping described herein which utilizesecreted individual MHC molecules have several advantages over the priorart, which utilized MHC from cells expressing multiple membrane-boundMHCs. While the prior art method could distinguish if an epitope waspresented on the surface of a cell, this prior art method is unable todirectly distinguish in which specific MHC molecule the peptide epitopewas bound. Lengthy purification processes might be used to try andobtain a single MHC molecule, but doing so limits the quantity andusefulness of the protein obtained. The novelty and flexibility of thecurrent invention is that individual MHC specificities can be utilizedin sufficient quantity through the use of recombinant, soluble MHCproteins.

[0042] Class I and class II MHC molecules are really a trimolecularcomplex consisting of an alpha chain, a beta chain, and the alpha/betachain's peptide cargo (i.e. peptide ligand) which is presented on thecell surface to immune effector cells. Since it is the peptide cargo,and not the MHC alpha and beta chains, which marks a cell as infected,tumorigenic, or diseased, there is a great need to identify andcharacterize the peptide ligands bound by particular MHC molecules. Forexample, characterization of such peptide ligands greatly aids indetermining how the peptides presented by a person with MHC-associateddiabetes differ from the peptides presented by the MHC moleculesassociated with resistance to diabetes. As stated above, having asufficient supply of an individual MHC molecule, and therefore that MHCmolecule's bound peptides, provides a means for studying such diseases.Because the method of the present invention provides quantities of MHCprotein previously unobtainable, unparalleled studies of MHC moleculesand their important peptide cargo can now be facilitated.

[0043] Therefore, the present invention is also related to methods ofepitope discovery and comparative ligand mapping which can be utilizedto distinguish infected/tumor cells from uninfected/non-tumor cells byunique epitopes presented by MHC molecules in the disease or non-diseasestate.

[0044] Creation of sHLA molecules from genomic DNA (gDNA)

[0045] 1. Genomic DNA Extraction. 200 μl of sample either blood, plasma,serum, buffy coat, body fluid or up to 5×10⁶ lymphocytes in 200 μlPhosphate buffered saline were used to extract genomic DNA using theQIAamp® DNA Blood Mini Kit blood and body fluid spin protocol. GenomicDNA quality and quantity was assessed using optical density readings at260 nm and 280 nm.

[0046] 2.1 PCR Strategy. Primers were designed for HLA-A, -B and -C lociin order to amplify a truncated version of the human class I MHC using a2 stage PCR strategy. The first stage PCR uses a primer set that amplifyfrom the 5′ Untranslated region to Intron 4. This amplicon is used as atemplate for the second PCR which results in a truncated version of theMHC Class I gene by utilizing a 3′ primer that sits down in exon 4, the5′ primer remains the same as the 1^(st) PCR. An overview can be seen inFIG. 1. The primers for each locus are listed in TABLE I. DifferentHLA-B locus alleles require primers with different restriction cut sitesdepending on the nucleotide sequence of the allele. Hence there are two5′ and two 3′ truncating primers for the -B locus. TABLE I Primer nameSequence 5′-3′ Locus Cut site Annealing site Seq ID NO. PP5UTAGCGCTCTAGACCCAGACGCCGAGGATGGCC A XbaI 5UT 1 3PPI4A GCCCTGACCCTGCTAAAGGTA Intron 4 2 PP5UTB GCGCTCTAGACCACCCGGACTCAGAATCTCCT B XbaI 5UT 3 3PPI4BTGCTTTCCCTGAGAAGAGAT B Intron 4 4 5UTB39 AGGCGAATTCCAGAGTCTCCTCAGACGCGB*39 EcoRI 5UT B39 5 5PKCE GGGCGAATTCCCGCCGCCACCATGCGGGTCATGGCGCC CEcoRI 5UT 6 3PPI4C TTCTGCTTTCCTGAGAAGAC C Intron 4 7 PP5UTGGGCGAATTCGGACTCAGAATCTCCCCAGACGCCGAG B EcoRI 5UT 8 PP3PEICCGCGAATTCTCATCTCAGGGTGAGGGGCT A, B, C EcoRI Exon 4 9 PP3PEIHCCGCAAGCTTTCATCTCAGGGTGAGGGGCT A, B, C HindIII Exon 4 10 3PEIHC7CCGCAAGCTTTCAGCTCAGGGTGAGGGGCT Cw*07 HindIII Exon 4 11

[0047] 2.2 Primary PCR. Materials: An Eppendorf Gradient Mastercycler isused for all PCR. (1) H₂O:Dionized ultrafiltered water (DIUF) FisherScientific, W2-4,41. (2) PCR nucleotide mix (10 mM eachdeoxyribonucleoside triphosphate [dNTP]), Boehringer Manheim, #1814,362. (3) 10× Pfx Amplification buffer, pH 9.0, GibcoBRL®, part # 52806,formulation is proprietary information. (4) 50 mM MgSO₄, GibcoBRL®, part#52044. (5) Platinum® Pfx DNA Polymerase (B Locus only), GibcoBRL®,11708-013. (6) Pfu DNA Polymerase (A and C Locus), Promega, M7741. (7)Pfu DNA Polymerase 10× reaction Buffer with MgSO₄, 200 mM Tris-HCL, pH8.8,100 mM KCl, 100 mM (NH₄)₂SO₄, 20 mM MgSO₄, 1 mg/ml nuclease freeBSA, 1% Triton®X-100. (8) Amplification primers (in ng/μl) (see TABLEI): A locus: 5′ sense PP5UTA (300); 3′ antisense PPI4A (300); B locus(Not B*39's): sense PP5UTB (300); antisense PPI4B (300); B locus(B*39's): sense 5UTB39 (300); antisense PPI4B (300); C Locus: sense5PKCE (300); antisense PPI4C (300). (9) gDNA Template.

[0048] 2.3 Secondary PCR (also used for colony PCR). (1) H₂O:Dionizedultrafiltered water (DIUF) Fisher Scientific, W2-4,41. (2) PCRnucleotide mix (10 mM each deoxyribonucleoside triphosphate [dNTP]),Boehringer Manheim, #1814, 362. (3) Pfu DNA Polymerase (A and C Locus),Promega, M7741. (4) Pfu DNA Polymerase 10× reaction Buffer with MgSO₄,200 mM Tris-HCL, pH 8.8,100 mM KCl, 100 mM (NH₄)₂SO₄, 20 mM MgSO₄, 1mg/ml nuclease free BSA, 1% Triton®X-100. (5) Amplification primers (inng/μl) see TABLE I: A-locus: 5′ sense PP5UTA (300), 3′ antisense PP3PEI(300); B-locus: sense PP5UTB (300), antisense PP3PEI (300); B-locus:sense PP5UT (300), antisense PP3PEIH (300); B-locus B39's: sense 5UTB39(300), antisense PP3PEIH (300); C-locus: sense 5PKCE (300), antisensePP3PEI (300); C-locus Cw*7's: sense 5PKCE (300), antisense 3PEIHC7(300). (6) Template 1:100 dilution of the primary PCR product.

[0049] 2.4 Gel Purification of PCR products and vectors. (1) Dark ReaderTansilluminator Model DR-45M, Clare Chemical Research. (2) SYBR Green,Molecular Probes Inc. (3) Quantum Prep Freeze 'N Squeeze DNA GelRxtraction Spin Columns, Bio-Rad Laboratories, 732-6165.

[0050] 2.5 Restriction digests, Ligation and Transformation. (1)Restriction enzymes from New England Biolabs: (a) EcoR I #R0101S; (b)Hind III #R0104S; (c) Xba I #R0145S. (2) T4 DNA Ligase, New EnglandBiolabs, #M0202S. (3) pcDNA3.1(−), Invitrogen Corporation, V795-20. (4)10× Buffers from New England Biolabs: (a) EcoR I buffer, 500 mM NaCl,1000 mM Tris-HCL, 10 mM MgCL₂, 0.25% Triton-X 100, pH 7.5; (b) T4 DNAligase buffer, 500 mM Tris-HCL, 100 mM MgCL₂, 100 mM DTT, 10 mM ATP, 250ug/ml BSA, pH 7.5; (c) NEB buffer 2,500 mM NaCl, 100 mM Tris-HCl, 100 mMMgCl₂, 10 mM DDT, pH 7.9. (5) 100× BSA, New England Biolabs. (6)Z-Competent E. coli Transformation Buffer Set, Zymo Research, T3002. (7)E. coli strain JM109. (8) LB Plates with 100 μg/ml ampicillin. (9) LBmedia with 100 μg/ml ampicillin.

[0051] 2.6 Plasmid Extraction. Wizard Plus SV minipreps, Promega,#A1460.

[0052] 2.7 Sequencing of Clones. (1) Thermo Sequenase Primer CycleSequencing Kit, Amersham Pharmacia Biotech, 25-2538-01. (2) CY5 labelledprimers (see TABLE II). (3) AlfExpress automated DNA sequencer, AmershamPharmacia Biotech. TABLE II Primer Name Sequence 5′-3′ Seq ID NO: T7PromTAATACGACTCACTATAGGG 12 BGHrev TAGAAGGCACAGTCGAGG 13 PPI2E2RGTCGTGACCTGCGCCCC 14 PPI2E2F TTTCATTTTCAGTTTAGGCCA 15 ABCI3E4FGGTGTCCTGTCCATTCTCA 16

[0053] 2.8 Gel Casting. (1) PagePlus 40% concentrate, Amresco, E562, 500ml. (2) Urea, Amersham Pharmacia Biotech, 17-0889-01,500g. (3) 3N′N′N′N′-tetramethylethyleneiamine (TEMED), APB. (4) Ammoniumpersulphate (10% solution), APB. (5) Boric acid, APB. (6) EDTA-disodiumsalt, APB. (7) Tris, APB. (8) Bind-Saline, APB. (9) Isopropanol, Sigma.(10) Glacial Acetic Acid, Fisher Biotech. (11) DIUF water, FisherScientific. (12) EtOH 200-proof.

[0054] 2.9 Plasmid Preparation for Electroporation. Qiagen Plasmid Midikit, Qiagen Inc., 12143.

[0055] 3.0 Electroporation. (1) Biorad Gene Pulser with capacitanceextender, Bio-Rad Laboratories. (2) Gene Pulser Cuvette, Bio-RadLaboratories. (3) Cytomix: 120 mM KCl, 0.15 mM CaCl₂, 10mMK₂HPO₄/KH₂PO₄, pH 7.6, 25 mM Hepes, pH 7.6, 2 mM EGTA, pH 7.6, 5 mMMgCl₂, pH 7.6 with KOH. (4) RPMI 1640+20% Foetal Calf Serum+Pen/strep.(5) Haemacytometer. (6) Light Microscope. (7) CO₂ 37° Incubator. (8)Cells in log phase.

[0056] Primary PCR A-Locus and C-Locus 10× Pfu buffer 5 μl 5′ Primer(300 ng/μl) 1 μl 3′ Primer (300 ng/μl) 1 μl dNTP's (10 mM each) 1 μlgDNA (50 ng/μl) 10 μl DIUF H₂O 31.4 μl Pfu DNA Polymerase 0.6 μl 96° C.2 min. 95° C. 1 min 58° C. 1 min {close oversize brace} ×35 73° C. 5 min73° C. 10 min B-locus 10× Pfx buffer 5 μl 5′ Primer (300 ng/μl) 1 μl 3′Primer (300 ng/μl) 1 μl dNTP's (10 mM each) 1.5 μl MgSO₄ (50 mM) 1 μlgDNA (100 ng/μl) 1 μl DIUF H₂O 40 μl Pfx DNA Polymerase 0.5 μl 94° C. 2min. 94° C. 1 min 60° C. 1 min {close oversize brace} ×35 68° C. 3.5 min68° C. 5 min

[0057] Gel Purification of PCR (all PCR and Plasmids are Gel Purified)

[0058] Mix primary PCR with 5 μl of 1× SYBR green and incubate at roomtemperature for 15 minutes then load on a 1% agarose gel. Visualize onthe Dark Reader and purify using the Quantum Prep Freeze and Squeezeextraction kit according to the manufacturers instructions.

[0059] Secondary PCR A, B and C Loci 10× Pfu buffer 5 μl 5′ Primer (300ng/μl) 0.5 μl 3′ Primer (300 ng/μl) 0.5 μl dNTP's (10 mM each) 1 μl1:100 1° PCR 10 μl DIUF H₂O 37.5 μl Pfu DNA Polymerase 0.5 μl 96° C. 2min. 95° C. 1 min 58° C. 1 min {close oversize brace} ×35 73° C. 4 min73° C. 7 min Restriction digests 2° PCR (gel purified) 30 μl Restrictionenzyme 1 × μl Restriction enzyme 2 × μl 10× buffer 5 μl 100× BSA 0.5 μlDIUF H₂O 10.5 μl

[0060] The enzymes used will be determined by the cut sites incorporatedinto the PCR primers for each individual PCR. The expression vectorpcDNA3.1 (−) will be cut in a similar manner.

[0061] Ligation PcDNA3.1 (-) cut with same enzymes as PCR 50 ng Cut PCR100 ng 10× T4 DNA ligase buffer 2 μl T4 DNA Ligase 1 μl DIUF H₂O up to20 μl

[0062] Transformation

[0063] Transform JM109 using competent cells made using Z-competent E.coli Transformation Kit and Buffer Set and follow the manufacturersinstructions.

[0064] Colony PCR

[0065] This will check for insert in any transformed cells. Follow thesame protocol for the secondary PCR.

[0066] Mini Preps of colonies with insert

[0067] Use the Wizard Plus SV minipreps and follow the manufacturersinstructions. Make glycerol stocks before beginning extraction protocol.

[0068] Sequencing of Positive Clones

[0069] Using the Thermo Sequenase Primer Cycle Sequencing Kit A, C, G orT mix 3 μl CY5 Primer 1 pm/μl 1 μl DNA template 100 ng/μl 5 μl 96° C. 2min 96° C. 30 sec {close oversize brace} ×25 61° C. 30 sec

[0070] Add 6 μl formamide loading buffer and load 10 μl onto sequencinggel. Analyse sequence for good clones with no misincorporations.

[0071] Midi Preps

[0072] Prepare plasmid for electroporation using the Qiagen Plasmid MidiKit according to the manufacturers instructions.

[0073] Electroporation

[0074] Electroporations are performed as described in “The Bw4 publicepitope of HLA-B molecules confers reactivity with natural killer cellclones that express NKb1, a putative HLA receptor. Gumperz, J. E., V.Litwin, J .H. Phillips, L. L. Lanier and P. Parham. J. Exp. Med.181:1133-1144, 1995, which is expressly incorporated herein byreference.”

[0075] Screening for Production of Soluble HLA

[0076] An ELISA is used to screen for the production of soluble HLA. Forbiochemical analysis, monomorphic monoclonal antibodies are particularlyuseful for identification of HLA locus products and their subtypes.

[0077] W6/32 is one of the most common monoclonal antibodies (mAb) usedto characterize human class I major histocompatibility complex (MHC)molecules. It is directed against monomorphic determinants on HLA-A, -Band -C HCs, which recognizes only mature complexed class I molecules andrecognizes a conformational epitope on the intact MHC moleculecontaining both beta2-microglobulin (β2m) and the heavy chain (HC).W6/32 binds a compact epitope on the class I molecule that includes bothresidue 3 of beta2m and residue 121 of the heavy chain (Ladasky J J,Shum B P, Canavez F, Seuanez H N, Parham P. Residue 3 ofbeta2-microglobulin affects binding of class I MHC molecules by theW6/32 antibody. Immunogenetics April 1999 ;49(4):312-20.). The constantportion of the molecule W6/32 binds to is recognized by CTLs and thuscan inhibit cytotoxicity. The reactivity of W6/32 is sensitive to theamino terminus of human beta2-microglobulin (Shields M J, Ribaudo R K.Mapping of the monoclonal antibody W6/32: sensitivity to the aminoterminus of beta2-microglobulin. Tissue Antigens May 1998;51(5):567-70). HLA-C could not be clearly identified inimmunoprecipitations with W6/32 suggesting that HLA-C locus products maybe associated only weakly with b2m, explaining some of the difficultiesencountered in biochemical studies of HLA-C antigens [Stam, 1986 #1].The polypeptides correlating with the C-locus products are recognizedfar better by HC-10 than by W6/32 which confirms that at least some ofthe C products may be associated with b2m more weakly than HLA-A and -B.W6/32 is available biotinylated (Serotec MCA81B) offering additionalvariations in ELISA procedures.

[0078] HC-10 is reactive with almost all HLA-B locus free heavy chains.The A2 heavy chains are only very weakly recognized by HC-10. Moreover,HC-10 reacts only with a few HLA-A locus heavy chains. In addition,HC-10 seems to react well with free heavy chains of HLA-C types. Noevidence for reactivity of HC-10 with heavy-chain/b2m complex has beenobtained. None of the immunoprecipitates obtained with HC-10 containedb2m [Stam, 1986 #1]. This indicates that HC-10 is directed against asite of the HLA class I heavy chain that includes the portion involvedin interaction with the β2m. The pattern of HC-10 precipitated materialis qualitatively different from that isolated with W6/32.

[0079] TP25.99 detects a determinant in the alpha3 domain of HLA-ABC. Itis found on denatured HLA-B (in Western) as well as partially or fullyfolded HLA-A,B,& C. It doesn't require a peptide or β2m, i.e. it workswith the alpha 3 domain which folds without peptide. This makes ituseful for HC determination.

[0080] Anti-human β2m (HRP) (DAKO P0174) recognizes denatured as well ascomplexed β2m. Although in principle anti-β2m reagents could be used forthe purpose of identification of HLA molecules, they are less suitablewhen association of heavy chain and β2m is weak. The patterns of class Imolecules precipitated with W6/32 and anti-β2m are usuallyindistinguishable [Vasilov, 1983 #10].

[0081] Rabbit anti-β2-microglobulin dissociates β2-microglobulin fromheavy chain as a consequence of binding (Rogers, M. J., Appella, E.,Pierotti, M. A., Invernizzi, G., and Parmiani, G. (1979) Proc Natl.Acad. Sci. U.S.A. 76, 1415-1419). It also has been reported that rabbitanti-human β2-microglobulin dissociactes β2-microglobulin from HLA heavychains upon binding (Nakamuro, K., Tanigaki, N., and Pressman, D. (1977)Immunology 32, 139-146.). This anti-human β2m antibody is also availableunconjugated (DAKO A0072).

[0082] The W6/32-HLA sandwich ELISA. Sandwich assays can be used tostudy a number of aspects of protein complexes. If antibodies areavailable to different components of a heteropolymer, a two-antibodyassay can be designed to test for the presence of the complex. Using avariation of these assays, monoclonal antibodies can be used to testwhether a given antigen is multimeric. If the same monoclonal antibodyis used for both the solid phase and the label, monomeric antigenscannot be detected. Such combinations, however, may detect multimericforms of the antigen. In these assays negative results may be generatedboth by multimeric antigen held in unfavorable steric positions as wellas by monomeric antigens.

[0083] The W6/32—anti-β2m antibody sandwich assay is one of the besttechniques for determining the presence and quantity of sHLA. Twoantibody sandwich assays are quick and accurate, and if a source of pureantigen is available, the assay can be used to determine the absoluteamounts of antigen in unknown samples. The assay requires two antibodiesthat bind to non-overlapping epitopes on the antigen. This assay isparticularly useful to study a number of aspects of protein complexes.

[0084] To detect the antigen (sHLA), the wells of microtiter plates arecoated with the specific (capture) antibody W6/32 followed by theincubation with test solutions containing antigen. Unbound antigen iswashed out and a different antigen-specific antibody (anti-β2m)conjugated to HRP is added, followed by another incubation. Unboundconjugate is washed out and substrate is added. After anotherincubation, the degree of substrate hydrolysis is measured. The amountof substrate hydrolyzed is proportional to the amount of antigen in thetest solution.

[0085] The major advantages of this technique are that the antigen doesnot need to be purified prior to use and that the assays are veryspecific. The sensitivity of the assay depends on 4 factors: (1) Thenumber of capture antibody; (2) The avidity of the capture antibody forthe antigen; (3) The avidity of the second antibody for the antigen; (4)The specific activity of the labeled second antibody.

[0086] Using an ELISA protocol template and label a clear 96-wellpolystyrene assay plate. Polystyrene is normally used as a microtiterplate. (Because it is not translucent, enzyme assays that will bequantitated by a plate reader should be performed in polystyrene and notPVC plates).

[0087] Coating of the W6/32 is performed in Tris buffered saline (TBS);pH 8.5 A coating solution of 8.0 μg/ml of specific W6/32 antibody in TBS(pH 8.5) is prepared. (blue tube preparation stored at −20° C. with aconcentration of 0.2 mg/ml and a volume of 1 ml giving 0.2 mg per tube).TABLE III No. of Total W6/32 TBS plates Volume antibody pH 8.5 1 10 ml 400 μl  9.6 ml 2 20 ml  800 μl 19.2 ml 3 30 ml 1200 μl 28.8 ml 4 40 ml1600 μl 38.4 ml 5 50 ml 2000 μl 48.0 ml

[0088] Although this is well above the capacity of a microtiter plate,the binding will occur more rapidly. Higher concentrations will speedthe binding of antigen to the polystyrene but the capacity of theplastic is only about 100 ng/well (300 ng/cm₂), so the extra proteinwill not bind. (If using W6/32 of unknown composition or concentration,first titrate the amount of standard antibody solution needed to coatthe plate versus a fixed, high concentration of labeled antigen. Plotthe values and select the lowest level that will yield a strong signal.Do not include sodium azide in any solutions when horseradish peroxidaseis used for detection.

[0089] Immediately coat the microtiter plate with 100 μl of antigensolution per well using a multichannel pipet. Standard polystyrene willbind antibodies or antigens when the proteins are simply incubated withthe plastic. The bonds that hold the proteins are non-covalent, but theexact types of interactions are not known. Shake the plate to ensurethat the antigen solution is evenly distributed over the bottom of eachwell. Seal the plate with plate sealers (sealplate adhesive sealingfilm, nonsterile, 100 per unit; Phenix; LMT-Seal-EX) or sealing tape toNunc-Immuno™ Modules (# 236366). Incubate at 4° C. overnight. Avoiddetergents and extraneous proteins. Next day, remove the contents of thewell by flicking the liquid into the sink or a suitable waste container.Remove last traces of solution by inverting the plate and blotting itagainst clean paper toweling. Complete removal of liquid at each step isessential for good performance.

[0090] Wash the plate 10 times with Wash Buffer (PBS containing 0.05%Tween-20) using a multi-channel ELISA washer. After the last wash,remove any remaining Wash Buffer by inverting the plate and blotting itagainst clean paper toweling. After the W6/32 is bound, the remainingsites on the plate must be saturated by incubating with blocking buffermade of 3% BSA in PBS. Fill the wells with 200 μl blocking buffer. Coverthe plates with an adhesive strip and incubate overnight at 4° C.Alternatively, incubate for at least 2 hours at room temperature whichis, however, not the standard procedure. Blocked plates may be storedfor at least 5 days at 4° C. Good pipetting practice is most importantto produce reliable quantitative results. The tips are just as importanta part of the system as the pipette itself. If they are of inferiorquality or do not fit exactly, even the best pipette cannot producesatisfactory results. The pipette working position is always vertical:Otherwise causing too much liquid to be drawn in. The immersion depthshould be only a few millimeters. Allow the pipetting button to retractgradually, observing the filling operation. There should be noturbulence developed in the tip, otherwise there is a risk of aerosolsbeing formed and gases coming out of solution.

[0091] When maximum levels of accuracy are stipulated, prewetting shouldbe used at all times. To do this, the required set volume is first drawnin one or two times using the same tip and then returned. Prewetting isabsolutely necessary on the more difficult liquids such as 3% BSA. Donot prewet, if your intention is to mix your pipetted sample thoroughlywith an already present solution. However, prewet only for volumesgreater than 10 μl. In the case of pipettes for volumes less than 10 μlthe residual liquid film is as a rule taken into account when designingand adjusting the instrument. The tips must be changed between eachindividual sample. With volumes <10 μl special attention must also bepaid to drawing in the liquid slowly, otherwise the sample will besignificantly warmed up by the frictional heat generated. Then slowlywithdraw the tip from the liquid, if necessary wiping off any dropsclinging to the outside.

[0092] To dispense the set volume hold the tip at a slight angle, pressit down uniformly as far as the first stop. In order to reduce theeffects of surface tension, the tip should be in contact with the sideof the container when the liquid is dispensed. After liquid has beendischarged with the metering stroke, a short pause is made to enable theliquid running down the inside of the tip to collect at its lower end.Then press it down swiftly to the second stop, in order to blow out thetip with the extended stroke with which the residual liquid can be blownout. In cases that are not problematic (e.g. aqueous solutions) thisbrings about a rapid and virtually complete discharge of the set volume.In more difficult cases, a slower discharge and a longer pause beforeactuating the extended stroke can help. To determine the absolute amountof antigen (sHLA), sample values are compared with those obtained usingknown amounts of pure unlabeled antigen in a standard curve.

[0093] For accurate quantitation, all samples have to be run intriplicate, and the standard antigen-dilution series should be includedon each plate. Pipetting should be preformed without delay to minimizedifferences in time of incubation between samples. All dilutions shouldbe done in blocking buffer. Thus, prepare a standard antigen-dilutionseries by successive dilutions of the homologous antigen stock in 3% BSAin PBS blocking buffer. In order to measure the amount of antigen in atest sample, the standard antigen-dilution series needs to span most ofthe dynamic range of binding. This range spans from 5 to 100 ng sHLA/ml.A stock solution Ê of 1 μg/ml should be prepared, aliquoted in volumesof 300 μl and stored at 4° C. Prepare a 50 ml batch of standard at thetime. (New batches need to be compared to the old batch before used inquantitation).

[0094] Use a tube of the standard stock solution Ê to prepare successivedilutions. While standard curves are necessary to accurately measure theamount of antigen in test samples, they are unnecessary for qualitative“yes/no” answers. For accurate quantitation, the test solutionscontaining sHLA should be assayed over a number of at least 4 dilutionsto assure to be within the range of the standard curve. Prepare serialdilutions of each antigen test solution in blocking buffer (3% BSA inPBS). After mixing, prepare all dilutions in disposable U-bottom 96 wellmicrotiter plates before adding them to the W6/32-coated plates with amultipipette. Add 150 μl in each well. To further proceed, remove anyremaining blocking buffer and wash the plate as described above. Theplates are now ready for sample addition. Add 100 μl of the sHLAcontaining test solutions and the standard antigen dilutions to theantibody-coated wells.

[0095] Cover the plates with an adhesive strip and incubate for exactly1 hour at room temperature. After incubation, remove the unbound antigenby washing the plate 10× with Wash Buffer (PBS containing 0.05%Tween-20) as described. Prepare the appropriate developing reagent todetect sHLA. Use the second specific antibody, anti-human β2m-HRP (DAKOP0174/0.4 mg/ml) conjugated to Horseradish Peroxidase (HRP). Dilute theanti-human β2m-HRP in a ratio of 1:1000 in 3% BSA in PBS. (Do notinclude sodium azide in solutions when horseradish peroxidase is usedfor detection). TABLE IV No. of Total anti-β2m-HRP 3% BSA plates Volumeantibody in PBS 1 10 ml 10 μl 10 ml 2 20 ml 20 μl  2 ml 3 30 ml 30 μl 30ml 4 40 ml 40 μl 40 ml 5 50 ml 50 μl 50 ml

[0096] Add 100 μl of the secondary antibody dilution to each well. Alldilutions should be done in blocking buffer. Cover with a new adhesivestrip and incubate for 20 minutes at room temperature. Prepare theappropriate amount of substrate prior to the wash step. Bring thesubstrate to room temperature.

[0097] OPD (o-Phenylenediamine) is a peroxidase substrate suitable foruse in ELISA procedures. The substrate produces a soluble end productthat is yellow in color. The OPD reaction is stopped with 3 N H₂SO₄,producing an orange-brown product and read at 492 nm. Prepare OPD freshfrom tablets (Sigma, P6787; 2 mg/tablet). The solid tablets areconvenient to use when small quantities of the substrate are required.After second antibody incubation, remove the unbound secondary reagentby washing the plate 10× with Wash Buffer (PBS containing 0.05%Tween-20). After the final wash, add 100 μl of the OPD substratesolution to each well and allow to develop at room temperature for 10minutes. Reagents of the developing system are light-sensitive, thus,avoid placing the plate in direct light. Prepare the 3 N H₂SO₄ stopsolution. After 10 minutes, add 100 μl of stop solution per 100 μl ofreaction mixture to each well. Gently tap the plate to ensure thoroughmixing.

[0098] Read the ELISA plate at a wavelength of 490 nm within a timeperiod of 15 minutes after stopping the reaction. The background shouldbe around 0.1. If your background is higher, you may have contaminatedthe substrate with a peroxidase. If the substrate background is low andthe background in your assay is high, this may be due to insufficientblocking. Finally analyze your readings. Prepare a standard curveconstructed from the data produced by serial dilutions of the standardantigen. To determine the absolute amount of antigen, compare thesevalues with those obtained from the standard curve.

[0099] Creation of Transfectants and Production of Soluble Class IMolecules

[0100] Transfectants were established as previously described(Prilliman, K R et al., Immunogenetics 45:379, 1997, which is expresslyincorporated herein by reference) with the following modifications: acDNA clone of B*1501 containing the entire coding region of the moleculewas PCR amplified in order to generate a construct devoid of thecytoplasmic domain using primers 5PXI(59-GGGCTCTAGAGGACTCAGAATCTCCCCAGAC GCCGAG-39) and 3PEI(59-CCGCGAATTCTCATCTCAGGGTGAG-39) as shown in TABLE V. Constructs werealso created containing a C-terminal epitope tag consisting of either 6consecutive histidines or the FLAG epitope(Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys). TABLE V Primers utilized to createB*1501-HIS and B*1501-FLAG were 5PXIa   n  d        3  P   E  I   H   I  S   (   5   9   -CCGCGAATTCTCAGTGGTGGTGGTGGTGGTGCCATCTCAGGGTGAG-39)  or3   P    E    I   F   L    A   G          (   5   9    -CCGCGAATTCTCACTTGTCATCGTCGTCCTTGTAATCCCATCTCAGGGTGAG-39).

[0101] PCR amplicons were purified using a Qiagen Spin PCR purificationkit (Qiagen, Levsden, The Netherlands) and cloned into the mammalianexpression vector pCDNA 3.1 (Invitrogen, Carlsbad, Calif., USA). TABLEV. After confirmation of insert integrity by bidirectional DNAsequencing, constructs were electroporated into the class I negativeB-lymphoblastoid cell line 721.221 (Prilliman, K R et al., 1997,previously incorporated herein by reference). Transfectants weremaintained in medium containing G418 post-electroporation and subclonedin order to isolate efficient producers of soluble class I as determinedby ELISA (Prilliman, K R et al, 1997, previously incorporated herein byreference). TABLE V Full- length or Seq. Primer name Sequence TruncatingNotes ID NO: HLA5UT GGGCGTCGACGGACTCAGAA either 5′ primer, Sal I cutsite 17 TCTCCCCAGACGCCGAG 5UTA GCGCGTCGACCCCAGACGCC either 5′ primer,Sal I cut site A-locus specific 18 GAGGATGGCC 5PXI GGGCTCTAGAGGACTCAGAAeither 5′ primer, Xba I cut site 19 TCTCCCCAGACGCCGAG CLSP23CCGCGTCGACTCAGATTCTC full-length 5′ primer, Sal I cut site C-locusspecific 20 CCCAGACGCCGAGATG LDC3UTA CCGCAAGCTTAGAAACAAAG full-length 3′primer, HindIII cut site A-locus specific 21 TCAGGGTT CLSP1085CCGCAAGCTTGGCAGCTGTC full-length 3′ primer, HindIII cut site C-locusspecific 22 TCAGGCTTTACAAG(CT)G 3UTA CCGCAAGCTTTTGGGGAGGG full-length 3′primer, HindIII cut site A-locus specific 23 AGCACAGGTCAGCGTGGGAA G 3UTBCCGCAAGCTTCTGGGGAGGA full-length 3′ primer, HindIII cut site B-locusspecific 24 AACATAGGTCAGCATGGGAAC 3PEI CCGCGAATTCTCATCTCAGG truncating3′ primer, EcoRI cut site 25 GTGAG 3PEIHIS CCGCGAATTCTCAGTGGTGGtruncating 3′ primer, EcoRI cut site adds hexa-histidine tail 26TGGTGGTGGTGCCATCTCAG GGTGAG 3PEIFLAG CCGCGATTCTCACTTGTCATC truncating 3′primer, EcoRI cut site adds FLAG-epitope 27 GTCGTCCTTGTAATCCCATCT5PKOZXB GGGCTCTAGACCGCCGCCAC either 5′ primer, Xba I cut site C-locusspecific 28 CATGCGGGTCATGGCGCC

[0102] Soluble B*1501, B*1501-HIS, and B*1501-FLAG were produced byculturing established transfectants in CP3000 hollow-fiber bioreactorsas previously described by Prilliman et al, 1997, which has previouslybeen incorporated herein by reference. Supernatants containing solubleclass I molecules were collected in bioreactor harvests and purified onW6/32 affinity columns. At least 2 column purifications were performedper molecule.

[0103] Ligand Purification, Edman Sequencing, and Reverse-phase HPLCSeparation of Peptides

[0104] Peptide ligands were purified from class I molecules by acidelution (Prilliman, K R et al., Immunogenetics 48:89, 1998 which isexpressly incorporated herein by reference) and further separated fromheavy and light chains by passage through a stirred cell (Millipore,Bedford, Mass., USA) equipped with a 3-Kd cutoff membrane (Millipore).Approximately {fraction (1/100)} volume of stirred cell flow throughcontaining peptide eluted from either B*1501, B*1501-HIS, or B*1501-FLAGwas subjected to 14 cycles of Edman degradation on a 492A pulsed liquidphase protein sequencer (Perkin-Elmer Applied Biosystems Division,Norwalk, Conn., USA) without the derivitization of cysteine. Edmanmotifs were derived by combining from multiple column elutions thepicomolar yields of each amino acid and then calculating the foldincrease over previous round as described in (Prilliman, K R et al,1998, previously incorporated herein by reference) and are shown in FIG.2.

[0105] Pooled peptide eluate was separated into fractions by RP-HPLC aspreviously described (Prilliman, K R et al, 1998, previouslyincorporated herein by reference). Briefly, 400-mg aliquots of peptideswere dissolved in 100 ml of 10% acetic acid and loaded onto a 2.1 3 150mm C18 column (Michrom Bioresources, Auburn, Calif., USA) using agradient of 2%-10% acetonitrile with 0.06% TFA for 0.02 min followed bya 10%-60% gradient of the same for 60 min. Fractions were collectedautomatically at 1-min intervals with a flow rate of 180 ml/min.

[0106] Mass Spectrometric Ligand Analysis

[0107] RP-HPLC fractions were speed-vacuumed to dryness andreconstituted in 40 ml 50% methanol, 0.5% acetic acid. Approximately 6ml from selected fractions were sprayed into an API-III triplequadrupole mass spectrometer (PE Sciex, Foster City, Calif., USA) usinga NanoES ionization source inlet (Protana, Odense, Denmark). Scans werecollected while using the following instrument settings:polarity—positive; needle voltage—1375 V; orifice voltage—65 V; N2curtain gas—0.6 ml/min; step size—0.2 amu; dwell time—1.5 ms; and massrange—325-1400. Total ion traces generated from each molecule werecompared visually in order to identify ions overlapping betweenmolecules. Following identification of ion matches, individual ions wereselected for MS/MS sequencing.

[0108] Sequences were predicted using the BioMultiView program (PESciex) algorithm predict sequence, and fragmentation patterns furtherassessed manually. Determinations of ion sequence homology to currentlycompiled sequences were performed using advanced BLAST searches againstthe nonredundant, human expressed sequence tag, and unfinished highthroughput genomic sequences nucleotide databases currently availablethrough the National Center for Biotechnology Information (NationalInstitutes of Health, Bethesda, Md., USA).

[0109] The methodology of the present invention provides a directcomparative analysis of peptide ligands eluted from class I HLAmolecules. In order to accomplish such comparative analyses,hollow-fiber bioreactors for class I ligand production were used alongwith reverse-phase HPLC for fractionating eluted ligands, and massspectrometry for the mapping and sequencing of peptide ligands. Theapplication of comparative ligand mapping also is applicable to celllines that express endogenous class I. Prior to peptide sequencedetermination in class I positive cell lines, the effects of adding aC-terminal epitope tag to transfected class I molecules was found tohave no deleterious effects. Either a tag consisting of 6 histidines(6-HIS) or a tag containing the epitope Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys(FLAG) was added to the C-terminus of soluble B*1501 through PCR. Theseconstructs were then transfected into class I negative 721.221 cells andpeptides purified as previously established (Prilliman, K R et al, 1998,previously incorporated herein by reference). Comparison of the twotailed transfectants with the untailed, soluble B*1501 allowed for thedetermination that tag addition had no effect on peptide bindingspecificity of the class I molecule and consequently had no deleteriouseffects on direct peptide ligand mapping and sequencing.

[0110] Edman Motifs

[0111] The most common means for discerning ligands presented by aparticular class I molecule is Edman sequencing the pool of peptideseluted from that molecule. In order to demonstrate that tailing class Imolecules with Cterminal tags does not disrupt endogenous peptideloading, Edman sequences of the peptide pools from B*1501, B*1501-HIS,and B*1501-FLAG was compared with previously published B*1501 data FIG.2. Motifs were assigned to each of the various B*1501 molecules as shownin FIG. 2. At the anchor position 2 (P2) a dominant Q and subdominant Mwas seen in motifs as previously published by Falk et al.(Immunogenetics 41:165, 1995) and Barber et al. (J Exp Med 184:735,1996). A more disparate P3 is seen in all molecules with F, K, N, P, R,and Y appearing; these results have also been previously reported byFalk and Barber. Again, a dominant Y and F are seen as the C-terminalanchors at P9 in all three molecules. The motif data for all threemolecules are in close accord, therefore, with the published standardmotifs.

[0112] Mass Spectrometric Profiles

[0113] Comparison of motifs for the surface bound, nontailed, and tailedB*1501 molecules identified no substantial differences in the pooledpeptides bound by the various forms of B*1501 tested. However, the aimof the present invention is to subtractively compare the individualpeptides bound by class I molecules from diseased and healthy cells.Subtractive analysis is accomplished through the comparison of massspectrometric ion maps and, as such, the ion maps of tailed and untailedclass I molecules were compared in order to determine the effect oftailing upon comparative peptide mapping.

[0114] Peptides derived from tailed and untailed B*1501 were separatedinto fractions via reverse phase HPLC (RP-HPLC). Each fraction was thenscanned using an API-III mass spectrometer in order to identify ionspresent in each fraction. Overall ion scans from RP-HPLC fractions 9,10, 11, 18, 19, and 20 were produced and visually compared in order toassess ions representing peptides overlapping between the threemolecules. FIG. 3. depicts a representative section of the ion mapsgenerated from each of the molecules. This comparison shows that thesame pattern of ions is produced by the different B*1501 moleculesanalyzed here. The manual comparison of ion maps from each of the threefractions found little to no difference in the peptides bound by each ofthe three molecules.

[0115] Ligand Sequences

[0116] After identification of ion matches in MS chromatograms of eachof the three molecules, individual ions were chosen for sequencing bytandem mass spectrometry in order to determine if ions were indeedmatched at the peptide-sequence level. Ten ions from each fraction wereinitially selected for MS/MS sequence generation. Fragmentation patternsfor each of the ions from each molecule were manually compared andidentical fragmentation patterns were counted as peptide-sequence levelmatches, as illustrated in FIG. 4. Of the peptide fragmentation patternsexamined, 52/57 (91%) were exact matches between the untailed moleculesand the 6-HIStailed protein (TABLE VI). A more disparate pattern offragmentation was identified in the FLAG-tailed ions selected for MS/MSsequencing: of the 57 ions selected for MS/MS fragmentation comparison,39 (70%) fragmentation patterns matched between the FLAG-tailed anduntailed molecules. Overall, 91 out of 113 (81%) spectra examined werein accord between the tailed molecules and soluble B*1501. TABLE VIPercent Molecules Ions Examined Ion Matches Matched B*1501-HIS 57 52 91%B*1501-FLAG 56 39 70% B*1501-Tagged 113  91 81%

[0117] Several ligand sequences were clearly determined from thefragmentation patterns produced. The ligand QGLISRGYSY, deriving fromhuman periplakin, was sequenced from those peptides eluted in fraction18. A second ligand, AVRDISEASVF, an 11-mer matching a span of the 40Sribosomal protein S26, was identified in fraction 20. Notably, these twopeptides lacked the strong consensus glutamine expected by the motifdata, a phenomenon previously reported by our laboratory when sequencingB*1501-eluted ligands (Prilliman, K R et al, 1997, previouslyincorporated herein by reference). Both these ligands, however,terminate with an aromatic tyrosine or phenylalanine; these amino acidswere both predicted to be strong anchors by Edman sequencing data and bypreviously published observations (Prilliman, K R et al, 1998,previously incorporated herein by reference).

[0118] One embodiment of the present invention contemplatescharacterizing peptide ligands bound by a given class I molecule bytransfecting that molecule into a class I negative cell line andaffinity purification of the class I molecule and bound peptide.Complications arise, however, when cell lines are chosen for study thatalready possess class I molecules. In this case, antibodies specific forone class I molecule must be used to selectively purify that class Imolecule from others expressed by the cell. Because allele-specificantibodies recognize epitopes in and around the peptide binding groove,variations in the peptides found in the groove can alter antibodyaffinity for the class I molecule (Solheim, J C et al., J Immunol151:5387, 1993; and Bluestone, J A et al., J Exp Med 176:1757, 1992).Altered antibody recognition can, in turn, bias the peptides availablefor elution and subsequent sequence analysis.

[0119] In order to selectively purify from a class I positive cell atransfected class I molecule and its peptide ligands in an unbiased way,it was necessary to alter the embodiment for class I purification in anon-class I positive cell. The C-terminal addition of a FLAG and 6-HIStag to a class I molecule that had already been extensivelycharacterized, B*1501 was shown to have little or no effect on peptidebinding. This methodology was designed to allow purification of a singleclass I specificity from a complex mixture of endogenously expressedclass I molecules. Ligands eluted from the tailed and untailed B*1501molecule were compared to assess the effect of a tail addition on thepeptide repertoire.

[0120] Pooled Edman sequencing is the commonly used method to determinethe binding fingerprint of a given molecule, and this methodology wasused to ascertain the large-scale effect of tail addition upon peptidebinding. We subjected {fraction (1/100)} of the peptides eluted fromeach class I MHC molecule to Edman degradation and derived motifs foreach of the molecules. Both the HIS- and FLAG-tailed motifs matchedpublished motifs for the soluble and membrane-bound B*1501. Each of themolecules exhibited motifs bearing a dominant P2 anchor of Q, a moredisparate P3 in which multiple residues could be found, and anotherdominant anchor of Y or F at P9. Small differences in the picomolaramounts of each of the amino acids detected during Edman sequencing havebeen noted previously in consecutive runs with the same molecule andmost likely reflect differences in cell handling and/or peptideisolation rather than disparities in bound peptides. Highly similarpeptide motifs indicated that the peptide binding capabilities of classI MHC molecules are not drastically altered by the addition of a tag.

[0121] In order to insure the ligands were not skewed after tagaddition, MS and MS/MS were used for the mapping and sequencing ofindividual peptides, respectively. Peptide mixtures subjected to MSprovided ion chromatograms (FIG. 3) that were used to compare the degreeof ion overlap between the three examined molecules. Extensive ionoverlap indicates that the peptides bound by these tailed and untailedB*1501 molecules were nearly identical.

[0122] Selected ions were then MS/MS sequenced in order to confirm thatmapped ion overlaps indeed represented exact ligand matches throughcomparison of fragmentation patterns between the three molecules (FIG.4). Approximately 60 peptides were chosen initially for MS/MS—ten fromeach fraction. Overall, fragmentation patterns were exact matches in amajority of the peptides examined (TABLE VI). Fragmentation patternscategorized as nonmatches resulted from a mixture of peptides present atthe same mass to charge ratio, one or more of which was present in thetagged molecule and not apparent in the spectra of the same ion fromB*1501. Of the sequence-level matches, ligands derived from HIS-tailedmolecules more closely matched those derived from B*1501 than thoseeluted from FLAG-tailed molecules. In total, 52/57 HIS peptides wereexact matches, whereas 39/56 FLAG peptides were equivalent. Thus, thedata indicates that the 6-HIS tag is less disruptive to endogenouspeptide binding than is the FLAG-tag, although neither tag drasticallyaltered the peptides bound by B*1501.

[0123] A handful of individual ligand sequences present in fractions ofpeptides eluted from all three molecules were determined by MS/MS. Thetwo clearest sequences, AVRDISEASVF and QGLISRGYSY, demonstrate thattailed class I molecules indeed load endogenous peptide ligands. Thissupports the hypothesis that addition of a C-terminal tag does notabrogate the ability of the soluble HLA-B*1501 molecule to naturallybind endogenous peptides. Further, both peptide sequences closelymatched those previously reported for B*1501 eluted peptides having adisparate N-terminus paired with a more conserved C-terminus consistingof either a phenylalanine or a tyrosine. Given the homologous Edmansequence, largely identical fragmentation patterns, and the peptideligands shared between the three molecules, we conclude that addition ofa C-terminal tag does not significantly alter the peptides bound byB*1501.

[0124] Mapping and subtractively comparing eluted peptides is a directmeans for identifying differences and similarities in the individualligands bound by a class I HLA molecule. Indeed, subtractive comparisonsdemonstrate how overlapping ligands bind across closely related HLA-B15subtypes as well as pointing out which ligands are unique tovirus-infected cells. Direct comparative analyses of eluted peptideligands is well suited for a number of purposes, not the least of whichis viral and cancer CTL epitope discovery. Addition of a C-terminalepitope tag provides a feasible method for production and purificationof class I molecules, and therefore, peptide ligands in cell linescapable of sustaining viral infection or harboring neoplastic agents, asillustrated in FIG. 5. Direct peptide analysis from such lines shouldyield important information on host control of pathogenic elements aswell as provide important building blocks for rational vaccinedevelopment.

[0125] The present invention further relates in particular to a novelmethod for detecting those peptide epitopes which distinguish theinfected/tumor cell from the uninfected/non-tumor cell. The resultsobtained from the present inventive methodology cannot be predicted orascertained indirectly; only with a direct epitope discovery method canthe unique epitopes described herein be identified. Furthermore, onlywith this direct approach can it be ascertained that the source proteinis degraded into potentially immunogenic peptide epitopes. Finally, thisunique approach provides a glimpse of which proteins are uniquely up anddown regulated in infected/tumor cells.

[0126] The utility of such HLA-presented peptide epitopes which mark theinfected/tumor cell are three-fold. First, diagnostics designed todetect a disease state (i.e., infection or cancer) can use epitopesunique to infected/tumor cells to ascertain the presence/absence of atumor/virus. Second, epitopes unique to infected/tumor cells representvaccine candidates. Here, we describe epitopes which arise on thesurface of cells infected with HIV. Such epitopes could not be predictedwithout natural virus infection and direct epitope discovery. Theepitopes detected are derived from proteins unique to virus infected andtumor cells. These epitopes can be used for virus/tumor vaccinedevelopment and virus/tumor diagnostics. Third, the process indicatesthat particular proteins unique to virus infected cells are found incompartments of the host cell they would otherwise not be found in.Thus, we identify uniquely upregulated or trafficked host proteins fordrug targeting to kill infected cells.

[0127] The present invention describes, in particular, peptide epitopesunique to HIV infected cells. Peptide epitopes unique to the HLAmolecules of HIV infected cells were identified by direct comparison toHLA peptide epitopes from uninfected cells.

[0128] As such, and only by example, the present method is shown to becapable of identifying: (1) HLA presented peptide epitopes, derived fromintracellular host proteins, that are unique to infected cells but notfound on uninfected cells, and (2) that the intracellularsource-proteins of the peptides are uniquely expressed/processed in HIVinfected cells such that peptide fragments of the proteins can bepresented by HLA on infected cells but not on uninfected cells.

[0129] The method of the present invnetion also, therefore, describesthe unique expression of proteins in infected cells or, alternatively,the unique trafficking and processing of normally expressed hostproteins such that peptide fragments thereof are presented by HLAmolecules on infected cells. These HLA presented peptide fragments ofintracellular proteins represent powerful alternatives for diagnosingvirus infected cells and for targeting infected cells for destruction(i.e., vaccine development).

[0130] A group of the host source-proteins for HLA presented peptideepitopes unique to HIV infected cells represent source-proteins that areuniquely expressed in cancerous cells. For example, through using themethodology of the present invention a peptide fragment ofreticulocalbin is uniquely found on HIV infected cells. A literaturesearch indicates that the reticulocalbin gene is uniquely upregulated incancer cells (breast cancer, liver cancer, colorectal cancer). Thus, theHLA presented peptide fragment of reticulocalbin which distinguishes HIVinfected cells from uninfected cells can be inferred to alsodifferentiate tumor cells from healthy non-tumor cells. Thus, HLApresented peptide fragments of host genes and gene products thatdistinguish the tumor cell and virus infected cell from healthy cellshave been directly identified. The epitope discovery method of thepresent invention is also capable of identifiying host proteins that areuniquely expressed or uniquely processed on virus infected or tumorcells. HLA presented peptide fragments of such uniquely expressed oruniquely processed proteins can be used as vaccine epitopes and asdiagnostic tools.

[0131] The methodology to target and detect virus infected cells may notbe to target the virus-derived peptides. Rather, the methodology of thepresent invention indicates that the way to distinguish infected cellsfrom healthy cells is through alterations in host encoded proteinexpression and processing. This is true for cancer as well as for virusinfected cells. The methodology according to the present inventionresults in data which indicates without reservation thatproteins/peptides distinguish virus/tumor cells from healthy cells.

[0132] Example of Comparative Ligand Mapping in Infected and UninfectedCells Creation of Soluble Class I Construct

[0133] EBV-transformed cell lines expressing alleles of interest(particularly A*0201, B*0702, and Cw*0702) were grown and class I HLAtyped through the sequenced-based-typing methodology described in Turneret al. 1998, J. Immunol, 161 (3) 1406-13) and U.S. Pat. No. 6,287,764Hildebrand et al. both of which are expressly incorporated herein intheir entirety by reference. Total RNA was 5pXI and 3pEI, producing aproduct lacking the cytoplasmic and transmembrane domains.Alternatively, a 3′ primer encoding a hexa-histidine or FLAG epitope tagwas placed on the C-terminus using the primers, 3pEIHIS or 3pEIFLAG(TABLE V). For the C-locus, a 5′ primer was used encoding the Kozakconsensus sequence. (Davis, et al. 1999. J. Exp. Med. 189: 1265-1274).Each construct was cut with the appropriate restriction endonculease(see TABLE V) and cloned into the mammalian expression vector pCDNA3.1-(Invitrogen, Carlsbad, Calif.) encoding either a resistance gene forG418 sulfate or Zeocin (Invitrogen).

[0134] Transfection in Sup-T1 cells. Sup-T1 T cells were cultured inRPMI 1640+20% fetal calf serum at 37° C. and 5% CO₂. Cells were splitdaily in order to maintain log-phase growth. Plasmid DNA was purifiedusing either Qiagen Midi-prep kits (Qiagen, Santa Clarita) or BioradQuantum Prep Midiprep Kit (Biorad, Hercules, Calif.) according to themanufacturer's protocol and resuspended in sterile DNAse-free water.Cells were electroporated with 30 μgs of plasmid DNA at a voltage of 400mV and a capacitance of 960 μF. Decay constants were monitoredthroughout electroporation and only transfections with decay times under25 mS were carried through to selection. Selection was performed on day4 post-transfection with 0.45 mg/mL Zeocin (Invitrogen) selective mediumcontaining 30% fetal calf with the pH adjusted visually to just higherthan neutral. Cells were resuspended in selective medium at 2×10^ 6cells per ml, fed until they no longer turned the wells yellow (usingthe pH indicated Phenol Red (Mediatech)), and allowed to sit until cellsbegan to divide. After the appearance of active division, cells wereslowly fed with selective medium until they reached medium (T-75) tissueculture flasks. Cells were then subcloned at limiting dilutions of 0.5,1, and 1.5 cells per well in 96-well tissue culture plates. Cells wereallowed to sit until well turned yellow, they were then gradually movedto 24 well plates and small (T-25) tissue culture flasks. Samples weretaken for soluble class I ELISA, and the best producers of class I werefrozen for later use at 5×10^ 6 cells/ml and stored at −135° C.

[0135] Soluble MHC class I ELISA. ELISAs were employed to test theconcentration of the MHC class I/peptide complexes in cell culturesupernatants. The monoclonal antibody W6/32 (ATCC, Manassas, Va.) wasused to coat 96-well Nunc Starwell Maxi-sorp plates (VWR, West Chester,Pa.). One hundred μls of test sample containing class I was loaded intoeach well of the plate. Detection was with anti-βB2 microglobulin (lightchain) antibody conjugated to horse-radish peroxidase followed byincubation with OPD (Sigma, St. Louis, Mo.). ELISA values were read by aSpectraMax 340 00A, Rom Version 2.04, February 1996, using the programSoftmax Pro Version 2.2.1 from Molecular Devices. For determination ofMHC class I complex in carboys prior to affinity purification (seebelow), each sample was tested in triplicate on at least 2 separateplates. Uninfected and infected harvest concentrations were read on thesame plate and uninfected samples were brought to 1% Triton X 100 priorto loading on the ELISA plate. This was in an attempt to minimizevariability in mass spectra generate due to large differences in theamount of peptide loaded onto affinity columns.

[0136] Full-length construct creation. Full-length constructs (in thepCDNA3.1-/G418 sulfate resistance vector) were created and transfectedinto the class I negative B-LCL 721.221 and T2. Both cell lines werecultured in RPMI-1640+10% fetal calf serum until growing at log phase.Cells were electroporated at 0.25 V and 960 μF capacitance. After 2days, the cells were pelleted and resuspended in selective mediumconsisting of RPMI-1640+20% FCS+1.5 mg/ml G418 sulfate (Mediatech,Herndon, Va.). Cells were treated in the same manner as above (Sup-T1transfection) after this point.

[0137] Cell pharm production. Eight liters of Sup-T1 soluble MHC class Itransfectants cultured in roller bottles in RPMI-1640+15% FCS+100 Upenicillin/streptomycin were centrifuged for 10 min at 1100× g.Supernatant was discarded and a total of 3×10^ 9 total cells wereresuspended in 200 mls of conditioned medium. Infected cells were thenadded to a feed bottle and inoculated through the ECS feed pump of aUnisyn CP2500 cell pharm (Unisyn, Hopkington, Mass.) into 30 kDmolecular-weight cut-off hollow-fiber bioreactors previously primed withRPMI-1640 containing 20% fetal calf serum. Cells were allowed toincubate overnight in the bioreactor at a temperature of 37° C. and at apH of 7.20 maintained automatically through CO₂ injection into themedium reservoir of the system. No new medium was introduced into thesystem during this time period and the ICS recirculation was maintainedat a low value of 400 mls/minute. ECS feed was begun 12 hours postinoculation at a rate of 100 mls/day with 15% FCS supplementedRPMI-1640; ICS feed was likewise begun at a rate of 1 L/day. ECSrecirculation was initiated at day 2 post-inoculation at a rate of 4L/day. ECS and ICS samples were taken at 24-hour intervals and sHLAELISAs (see above) and glucose tests performed. ECS and ICS feed ratesas well as ECS and ICS recirculation rates were adjusted based onincreasing concentrations of sHLA in the harvest and decreasing levelsof glucose in the ICS medium.

[0138] Virus production and infection HIV MN-1 production. HIV MN-1cloned virus (Genbank Accession Number M17449) was thawed from frozenstock and used to infect 25×10^ 6 non-transfected Sup-T1 (Denny C T, et.al. 1986. Nature. 320:549.51, which is expressly incorporated herein inits entirety by reference) T cells using standard methods. Cells werecultured in RPMI-1640+20% fetal bovine serum (MediaTech) for 5 days andobserved for syncitia formation. Upon formation of syncitia, new cellswere added in fresh RPMI-1640/20% FCS. Culture was continued for 5 moredays when 100 mls of infected cells were removed. Supernatant was passedthrough a 0.45 um filter and cell-free virus was aliquotted and storedat −80° C. This process was continued until an appropriate amount ofvirus was harvested.

[0139] HIV-1 NL4-3 production. The infectious molecular clone pNL4-3(Genbank Accession Number AF324493) was transformed into the Esherichiacoli strain Top10F′ (Invitrogen, Carlsbad, Calif.). Plasmid DNA wasmidiprepped from transformed cells using either the Qiagen Midi Prep Kit(Qiagen, Santa Clarita, Calif.) or the Biorad Quantum Prep Midiprep Kit(Biorad, Hercules, Ca) according to the manufacturer's instructions.Plasmid DNA was used to transfect 293T cells (GenHunter Corporation,Nashville, Tenn.) using Roche's Fugene 6 reagent (Roche, Basel,Switzerland) following the manufacturer's protocol. Virus-containingsupernatant was harvested at 24, 48, and 72 hours, clarified bycentrifugation at 500× g for 10 min, aliquotted, and stored at −80° C.Sup-T1 transfectants containing either soluble A*0201, B*0702, orCw*0702 were cocultured with virus resulting in high-titre virus. After72 hours, infected cells were centrifuged at 1100× g for 10 minutes.Supernatant containing cell-free virus was removed, passed through a0.45 μm filter, aliquotted, and stored at −80° C. Virally-infected cellswere resuspended in freeze medium (RPMI-1640+20% FCS+10% DMSO) atapproximately 6×10^ 6 cells per ml and stored at −80° C.

[0140] Viral Titer Determination. One vial of frozen viral stock derivedfrom either strain of HIV was thawed and used in a TCID₅₀ assay scoredtwo ways: 1) wells containing at least 3 syncitia were consideredpositive or 2) wells containing over 50 ng/ml p24 antigen as determinedby ELISA were considered positive. The TCID₅₀ was then calculated usingthe Spearman-Karber method (DAIDS Virology Manual for HIV Laboratories,January 1997). The average of both scoring methods was used as the finaltiter of the virus. As a second means of viral titer monitoring, viralstock was used undiluted in a p24 ELISA (Beckman Coulter, Miami, Fla.)in order to determine the ngs of p24 present in cell-free virus.

[0141] P24 ELISA. Determination of HIV p24 major core protein wasdetermined by the commercially available Beckman Coulter p24 ELISAaccording to the manufacturer's instructions with the exceptions of thefollowing modifications: samples were treated with 10% Triton-X 100prior to removal from a BSL-3 facility, therefore the inactivationmedium included in the kit was not used. Secondly, samples were seriallydiluted in water prior to use.

[0142] Hollow-fiber bioreactor culture of infected cells. All workincluding large-scale culture of HIV was performed in a Biosafety Level3 Laboratory in accordance with guidelines set forth by the NationalInstitutes of Health. HIV MN-1 frozen viral stock aliquots were thawedand pooled to a 100 ml total volume, containing approximately 5.5×10^ 6TCID₅₀'s. Eight liters of Sup-T1 soluble MHC class I transfectantscultured in roller bottles in RPMI-1640+15% FCS+100 Upenicillin/streptomycin were centrifuged for 10 min at 1100× g.Supernatant was discarded and a total of 3×10^ 9 total cells wereresuspended in 200 mls of conditioned medium. The 100 mls of cell-freeHIV MN-1 was then added to the resuspended cells and incubated at 37° C.in %5 CO₂ for 2 hours with gentle shaking every 20 minutes. Infectedcells were then added to a feed bottle and inoculated through the ECSfeed pump of a Unisyn CP2500 cell pharm (Unisyn, Hopkington, Mass.)into: 30 kD molecular-weight cut-off hollow-fiber bioreactors previouslyprimed with RPMI-1640 containing 20% fetal calf serum. Cells wereallowed to incubate overnight in the bioreactor at a temperature of 37°C. and at a pH of 7.20 maintained automatically through CO₂ injectioninto the medium reservoir of the system. No new medium was introducedinto the system during this time period and the recirculation wasmaintained at a low value of 400 mls/minute. ECS feed was begun 12 hourspost inoculation at a rate of 100 mls/day with 15% FCS supplementedRPMI-1640; ICS feed was likewise begun at a rate of 1 L/day. ECS and ICSsamples were taken at 24-hour intervals, inactivated by addition ofTriton-X 100 to 1%, and sHLA ELISAs, p24 ELISAs, and glucose testsperformed as described above. ECS and ICS feed rates as well as ECS andICS recirculation rates were adjusted based on increasing concentrationsof sHLA in the harvest and decreasing levels of glucose in the ICSmedium.

[0143] Soluble HLA purification. Soluble-HLA containing supernatant wasremoved in 1.9 L volumes from infected hollow-fiber bioreactors.Twenty-percent Triton-X 100 was sterilized and placed in 50 ml aliquotsin 60 mls syringes; 2 syringes were injected into each 1.9 L harvestbottle as it was removed from the cell pharm, resulting in a final TX100 percentage of 1%. Bottles were inverted gently several times to mixthe TX 100 and stored at 4° C. for a minimum of 1 week. After 1 week,harvest was centrifuged at 2000× g for 10 minutes to remove cellulardebris and pooled into 10 L carboys. An aliquot was then removed fromthe pooled, HIV-inactivated supernatant and used in a quantitativeTCID₅₀ assay (as described above) and used to initiate a coculture withSup-T1's. Only after demonstration of a completely negative coculture aswell as TCID₅₀ were harvests removed from the BSL-3.

[0144] Class I/Peptide Production and Peptide Characterization Handlingof MHC classI/peptide complexes from infected cells. Each 10 L of cellpharm harvest was separated strictly on a temporal basis during the cellpharm run. (This was an attempt to assess any epitopic changes thatmight occur temporally during infection as opposed to those that mightoccur more globally.) Harvest was treated exactly as described above,except for the removal of a 2 ml aliquot for tests in both a TCID₅₀assay and cell coculture assay to determine infectivity of the virus.

[0145] Affinity purification of infected and uninfected MHC class Icomplexes. Uninfected and infected harvest removed from CP2500 machineswere treated in an identical manner post-removal from the cell pharm.Approximately 50 mgs total class I as measured by W6/32 ELISA (seeabove) were passed over a Pharmacia XK-50 (Amersham-Pharmacia Biotech,Piscataway, N.J.) column packed with 50 mls Sepharose Fast Flow 4Bmatrix (Amersham) coupled to W6/32 antibody. Bound class I complexeswere washed first with 1 L 20 mM sodium phosphate wash buffer, followedby a wash with buffer containing the zwitterionic detergent Zwittergent3-08 (Calbiochem, Merck KgaA, Darmstadt, Germany) at a concentration of10 mM, plus NaCl at 50 mM, and 20 mM sodium phosphate. The zwittergentwash was monitored by UV absorption at a wavelength of 216 nm forremoval of Triton-X 100 hydrophobically bound to the peptide complexes.After 1 L of wash had passed over the column (more than a sufficientamount for the UV to return to baseline), zwittergent buffer was removedwith 2 L of 20 mM sodium phosphate wash buffer. Peptides were elutedpost wash with freshly made 0.2N acetic acid, pH 2.7.

[0146] Peptide isolation and separation. Post-elution,peptide-containing eluate fractions were brought up to 10% glacialacetic acid concentration through addition of 100% glacial acetic acid.Fractions were then pooled into a model 8050 stirred cell (Millipore,Bedford, Mass.) ultrafiltration device containing a 3 kDmolecular-weight cutoff regenerated cellulose membrane (Millipore). Thedevice was capped and tubing parafilmed to prevent leaks and placed in a78° C. water bath for 10 minutes. Post-removal, the peptide-containingelution buffer was allowed to cool to room temperature. The stirred cellwas operated at a pressure of 55 psi under nitrogen flow. Peptides werecollected in 50 ml conical centrifuge tubes (VWR, West Chester, Pa.),flash frozen in super-cooled ethanol, and lyophilized to dryness.Peptides were resuspended either in 10% acetic acid or 10% acetonitrile.Peptides were purified through a first-round of HPLC on a Haisil C-18column (Higgins Analytical, Moutain View, Calif.), with an isocraticflow of 100% B (100% acetonitrile, 0.01% TFA) for 40 minutes. Followingelution, peptide-containing fractions were pooled, speed-vacuumed todryness, and resuspended in 150 μls of 10% acetic acid. Two μgs of thebase methyl violet were added to the peptide mixture in 10% acetic acidand this was loaded onto a Haisil C-18 column for fractionation.Peptides were fractionated by one of two methods, the latter resultingin increased peptide resolution. The first fractionation program was2-10% B in 2 minutes, 10-60% B in 60 minutes, with 1 minute fractioncollection. The second RP-HPLC gradient consisted of a 2-14% B in 2minutes, 14-40% B in 60 minutes, 40-70% B in 20 minutes, with 1 minutefraction collection. Peptides eluting in a given fraction were monitoredby UV absorbance at 216 nm. Separate but identical (down to the samebuffer preparations) peptide purifications were done for eachpeptide-batch from uninfected and infected cells.

[0147] Mass-spectrometric mapping of fractionated peptides. Fractionatedpeptides were mapped by mass spectrometry to generate fraction-based ionmaps. Fractions were speed-vacuumed to dryness and resuspended in 12 μls50:50 methanol:water+0.05% acetic acid. Two μls were removed and sprayedvia nanoelectrospray (Protana, Odense, Denmark) into a Q-Star quadrupolemass spectrometer with a time-of-flight detector (Perseptive SCIEX,Foster City, Calif.). Spectra were generated for masses in the range of50-1200amu using identical mass spectrometer settings for each fractionsprayed. Spectra were then base-line subtracted and analyzed using theprograms BioMultiview version 1.5beta9 (Persceptive SCIEX) or BioAnalystversion 1.0 (Persceptive SCIEX). Spectra from the same fraction inuninfected/infected cells were manually aligned to the same mass range,locked, and 15 amu increments visually assessed for the presence ofdifferences in the ions represented by the spectra (for an example, seeHickman et al. 2000. Human Immunology. 61:1339-1346 which is expresslyincorporated herein by reference). Ions were selected for MS/MSsequencing based on upregulations or downregulation of 1.5 fold over thesame ion in the uninfected cells, or the presence or absence of the ionin infected cells. Ions were thus categorized into multiple categoriesprior to MS/MS sequencing.

[0148] Tandem mass-spectrometric analysis of selected peptides. Lists ofions masses corresponding to each of the following categories weregenerated: 1) upregulated in infected cells, 2) downregulated ininfected cells, 3) present only in infected cells, 4) absent in infectedcells, and 5) no change in infected cells. The last category wasgenerally disregarded for MS/MS analysis and the first 4 categories weresubjected to MS/MS sequencing on the Q-Star mass spectrometer.Peptide-containing fractions were sprayed into the mass spectrometer in3 μl aliquots. All MS settings were kept constant except for the Q0 andCad gas settings, which were varied to achieve the best fragmentation.Fragmentation patterns generated were interpreted manually and with theaid of BioMultiView version 1.5 beta 9. No sequencing algorithms wereused for interpretation of data, however multiple web-based applicationswere employed to aid in peptide identification including: MASCOT(Perkins, D N et al. 1999. Electrophoresis. 20(18):3551-3567), ProteinProspector (Clauser K. R. et al. 1999. Analytical Chemistry. 71:2871),PeptideSearch(http://www.narrador.emblheidelberg.de/GroupPages/PageLink/Peptidesearchpage.html)and BLAST search (http://www.ncbi.nim.nih.gov/BLAST/).

[0149] Quality control of epitope changes. Multiple parameters wereestablished before peptides identified in the above fashion were deemed“upregulated,” “downregulated,” etc. First, the peptide fractions beforeand after the fraction in which the peptide was identified weresubjected to MS/MS at the same amu under the identical collisionconditions employed in fragmentation of the peptide-of-interest and thespectra generated overlaid and compared. This was done to make surethat, in the unlikely event that the peptides had fractionateddifferently (even with methyl-violet base B standardization) there wasnot the presence of the peptide in an earlier or later fraction of theuninfected or infected peptides (and that the peptides had trulyfractionated in an identical manner.) Secondly, the same amu that wasused to identify the first peptide was then subjected to MS/MS in thealternate fraction (either infected or uninfected, whichever wasopposite of the fraction in which the peptide was identified.) Spectraagain were overlaid in order to prove conclusively that thefragmentation patterns did not match and thus the peptide was notpresent in the uninfected cells;, or, in the case that the fragmentationpatterns did match, that the peptides were upregulated in the infectedcells. Finally, synthetic peptides were generated for each peptideidentified. These peptides were resuspended in 10% acetic acid andRP-HPLC fractionated under the same conditions as employed for theoriginal fractionation, ensuring that the peptide putatively identifiedhad the same hydrophobicity as that of the ion MS/MS fragmented. Thissynthetic peptide was MS/MS fragmented under the same collisionconditions as that of the ion, the spectra overlaid, and checked for anexact match with the original peptide fragment.

[0150] Functional Analysis\Literature Searches. After identification ofepitopes, literature searches were performed on source proteins todetermine their function within the infected cell. Broad inferences canbe made from the function of the protein. Source proteins wereclassified into groups according to functions inside the cell. Again,broad inferences can be made as to the groups of proteins that would beavailable for specific presentation solely on infected cells. Secondly,source proteins were scanned for other possible epitopes which may bebound by other MHC class I alleles. Peptide binding predictions (Parker,K. C., et. al. 1994. J. Immunol. 152:163) were employed to determine ifother peptides presented from the source proteins were predicted tobind. Proteasomal prediction algorithms (A. K. Nussbaum, et. Al. 2001.Immunogenetics 53:87-94) were likewise employed to determine thelikelihood of a peptide being created by the proteasome.

[0151] Sequence Identification. A discussion of the results seen withthe application of this procedure is included using the peptideGPRTAALGLL as an example. Other examples and data obtained based on themethodology are listed in TABLE VII. TABLE VII ACCESS- SEQ ID IONFRACTION SEQUENCE MW OBS′D MW SOURCE PROTEIN START AA ION # CATEGORY NO:Peptides Identified on Infected cells that are not present on UninfectedCells 612.720 32INF EQMFEDIISL 1223.582 1223.418 HIV MN-1, ENV 101 HIV-29 DERIVED 509.680 31INF IPCLLISFL 1017.601 1017.334 CHOLINERGICRECEPTOR, 250 30 ALPHA-3 POLYPEPTIDE 469.180 31INF STTAICATGL 936.466936.360 UBIQUITIN-SPECIFIC 152 10720340 31 PROTEASE 420.130 16INFAPAQNPEL 838.426 838.259 B-ASSOCIATED TRANSCRIPT PROTEIN 3 (BAT3) MHCGENE 32 PRODUCT 500.190 28INF LVMAPRTVL 998.602 998.396 HLA-B HEAVYCHAIN LEADER 2 4566550 MHC GENE 33 SEQUENCE PRODUCT 529.680 31INFAPFI[NS]PADX 1057.388 UNKNOWN, CLOSE TO SEVERAL cDNA's UNKNOWN 34523.166 12INF TPQSNRPVm 1044.500 1044.333 RNA POLYMERASE II 527 4505939RNA 35 POLYPEPTIDE A MACHIN- ERY/ BINDING PR 444.140 16INF AARPATSTL887.495 887.280 EUK, TRANSLATION 1073 Q04637 RNA 36 INITIATION FACTOR 4MACHIN- ERY/ BINDING PR 470.650 16INF MAMMAALMA 940.413 939.410SPARC-LIKE PROTEIN 19 478522 TUMOR 37 SUP- PRESSOR GENE 490.620 16INFIATVDSYVI 979.240 TENASCIN-C (HEXABRACHION) 1823 13639246 TUMOR 38 SUP-PRESSOR GENE 563.640 16INF SPNQARAQAAL 1126.597 1126.364 POLYPYRIMIDINETRACT- 141 131528 RNA 39 BINDING PROTEIN 1 MACHIN- ERY/ BINDING PR 30INFGPRTAALGLL 968.589 968.426 RETICULOCALBIN 4 4506457 TUMOR 40 SUP-PRESSOR GENE 556.150 16INF NPNQNKNVAL 11111.586 1111.300 ELAV (HuR) 1884503551 RNA 41 MACHIN- ERY/ BINDING PR Peptides Identified on Uninfectedcells that are not present on Infected cells 16UNINF GSHSMRY MHC CLASS IHEAVY CHAIN variable multiple MHC 42 (could derive from Class I multiplealleles, Product i.e., HLA-B* 0702 OR HLA-G, etc.)

[0152] The first step in identification of an epitope present only onuninfected cells is performing MS ion mapping. In this case, thereversed-phase HPLC fraction 30 obtained from HIV as disclosedhereinabove (which contains a fraction of the total class I peptides)was sprayed into the mass spectrometer and an ion spectrum created. FIG.6 shows the sections of ion map in which an ion was first identified asupregulated. The ion at 484.74 can be seen to predominate in the uppermap, which is the spectrum generated from peptides from the infectedcells. One can also see that there are other peptides which differ intheir intensities between the uninfected cells from one spectrum toanother. After a peptide is initially identified, the area of thespectrum in which the peptide is found is zoomed in on in order to morefully see all the ions in the immediate area (FIG. 7). After zooming inon the area from 482-488 amu, the ion at 484.72 can be seen to only bepresent in the infected cells (which are seen in the spectrum on thetop). A large difference such as this is not always seen, sometimes moreminor differences are chosen for sequence determination. This ion,however, was considered an extremely good candidate for furtheranalysis.

[0153] After identification of the ion, the next step in the process isto sequence the peptide by using tandem mass spectrometry. FIG. 8 showsthe spectrum generated when the peptide is fragmented. These fragmentsare used to discern the amino acid sequence of the peptide. The sequenceof this peptide was determined to be GPRTAALGLL. This peptide wasisolated from infected HLA-B*0702 molecules. One early quality controlstep is examining the peptide's sequence to see if it fits the sequencesthat were previously shown to be presented by this molecule. B*0702binds peptides that have a G at their second position (P2) and an L astheir C-terminal anchor. Based on this information, this sequence islikely to be a peptide presented by B*0702.

[0154] Descriptive characterization of peptide. Once the peptidesequence is obtained, information is gained on the source protein fromwhich the peptide was derived in the cytosol of the infected cell.Initially, a BLAST search (available at the National Center forBiotechnology website) is done to provide protein information on thepeptide. A BLAST search with the sequence GPRTAALGLL pulled up theprotein reticulocalbin 2. After the source protein is known, informationabout the protein is ascertained first from the PubMed (again availableat the National Center for Biotechnology website) and put into a formatto which one can easily refer as seen in FIG. 9. All of the accessionnumbers for the protein, as well as the original description of theprotein are included. This makes it easy to come back to the informationfor downstream use. Also, the protein sequence is copied, pasted, andsaved as a text document for incorporation into later searches. Thepeptide is highlighted in the entire protein, giving some context as towhere it is derived and how large the total protein is. This is theinitial data gathering step post-sequence determination.

[0155] The next step in characterizing the ligand is doing literaturesearches on the source protein from which the peptide was derived. Theprotein is entered into the PubMed database and all entries with theword “reticulocalbin” are retrieved. FIG. 10 illustrates the listingthat is done to summarize what has previously been described for thisprotein. It can be seen that for reticulocalbin, multiple articles havebeen published involving this protein. The literature is summarized in aparagraph following the PubMed listings and put into the report. Forreticulocalbin, some of the most interesting points are that it is an ERresident protein, which can lead to speculation on why it is presentedon infected cells. Secondly, it has been previously found to beupregulated in several other types of cancers, such as breast andcolorectal cancers. This again leads to speculation that this proteinmay be broadly applicable to treat more maladies than those caused byHIV. It is also determined whether or not this protein has beenpreviously cited as interacting with/or being interfered with by HIV.This was not seen for reticulocalbin and thus was not listed in thereport, (although in some instances it is seen.) A broad understandingof the protein is gained through literature searches.

[0156] Predictive characterization of peptide. After the literaturesearch, several secondary searches are performed. FIG. 11 illustratesthe results of a peptide-binding algorithm performed using Parker'sPrediction (which is described hereinabove). The entire source proteinis used for input and the computer generates a list of peptides whichare bound by the HLA allele chosen. In this case, B*0702 was chosenbecause that was the allele from which this peptide was derived. Fromthe black arrow in the figure, it can be seen that the peptide sequencedby mass spectrometry is predicted to bind to HLA-B*0702 with a highaffinity. Several other peptides are listed that are predicted to bindas well. FIG. 12 shows the same procedure being performed with thesource peptide using another well-known search engine, SYPEITHI. (Thisengine can be found on the worldwide web using the URL:http://syfpeithi.bmi-heidelberg.com/Scripts/MHCServer.dll/EpPredict.htm.)Again, the results from this search engine for B*0702 shows that thispeptide is predicted to bind to HLA-B*0702 with a high affinity. Also,multiple other peptides are predicted to be derived from this sourceprotein and bound. This prediction allows us to determine severalthings. First, we can tell if the peptide is predicted to be bound byprevious algorithms. This allows us to know how well the programs work,and/or if other people could identify this peptide (if they had thesource protein) from peptide binding algorithms. All of this informationcan be translated into increasing importance for the present inventivemethodology not only for the peptide but also for the source proteinitself.

[0157] After peptide-binding algorithms are performed, searches are doneto determine whether the peptides would be created by the proteasomeduring normal processing of proteins into peptides. It should bestrongly noted that multiple pathways for class I peptide loading arenow being demonstrated and that the cleavage algorithms for humanproteasomes are not well established by any means. While a positiveresult may indicate that the proteasome is largely responsible forcleavage, a negative result by no means indicates that the peptide isnot presented in the class I molecule. FIG. 13 shows the results of thefirst proteasomal cleavage done for the source protein reticulocalbinusing the cleavage predictor PaProC (available at URLhttp://www.paproc.de/). The epitope is outlined. By this predictionsoftware, the peptide is not predicted to be cleaved by the normalproteasome. This may mean that in infected cells, alternative pathwaysof MHC class I presentation are being used, particularly in reference tothe reticulocalbin peptide. This, in turn, may present novel methods fortherapeutics during viral infection. A second proteasomal cleavagesearch is also employed using the prediction software NetChop (availableon the worldwide web) as seen in FIG. 14. By this prediction and otherdata from current literature in the field, the peptide would be createdby the proteasome and cleaved to form the GPRTAALGLL identified.

[0158] A third round of analysis involves only the source protein. Allother alleles are tested for peptide binding and lists of the highestbinders generated. The proteasomal cleavage predictions are thenreferred to in order to elucidate how these peptides are generated. Thisinformation is useful for downstream testing of peptides and fordetermining whether or not this protein will be applicable for vaccinetrials covering a broad range of HLA alleles. For reticulocalbin,multiple high-affinity peptides were demonstrated for differing HLAalleles (some examples of which are shown in FIG. 15) In this figure,several high affinity peptides deriving from reticulocalbin wereidentified for HLA-A*0201 and A*0101.

[0159] Quality control of sequence determination. There currently existsno direct means to score the quality of MS/MS sequence data. Once alldescriptive and predictive steps are concluded, we return again to theoriginal peptide sequence for quality control to ensure that the peptideis indeed what we have identified as the amino acid sequence and thatthe peptide is truly present only in infected cells. We employ thesemultiple steps so there is no doubt that the sequence is truly what weclaim it to be before we move on to downstream applications involvingthe peptide.

[0160] Initially, we determine that the peptide is truly upregulated orpresent only in infected cells. For the reticulocalbin peptide, wedetermined that this peptide was probably only present in infectedcells. In order to make certain that the peptide was truly absent in theuninfected cells and that there was no chance that our RP-HPLCfractionation had differed (remembering that we use internal controlsfor our fractionation as well) we generated ion spectra using MS fromthe fractions before and after the one in which we identified thepeptide. In the case of the reticulocalbin peptide, we identified thepeptide in fraction 30, so we performed MS on fractions 29 and 31 (FIG.16) In FIG. 16, it can be seen that there is no substantial peak at them/z 484.72. This indicated that there was not differential fractionationand that the peptide truly was absent from uninfected cells. In the casethat there was a peptide peak in one of the before or after fractions,we would then turn to MS/MS to determine whether this peak representedthe ion we were characterizing or another ion with the samemass-to-charge ratio.

[0161] After determining that the peptide is not present in anotherfraction, MS/MS was preformed on the same m/z in the uninfected spectrum(in the same fraction) in order to conclusively prove that there is nopeptide present with the same sequence in the uninfected cells. In FIG.17 one can see that the fragmentation patterns produced under identicalMS collision conditions are totally different. This illustrates theabsence of the reticulocalbin peptide in the uninfected cells.

[0162] Finally, in order to conclusively prove that the peptide sequenceis the same as that originally identified, we synthesize syntheticpeptides consisting of the same amino acids as the peptide sequenceidentified from the MS/MS fragmentation pattern. For the reticulocalbinpeptide (i.e. the ion in fraction 30 at 484.72) we synthesized thepeptide “GPRTAALGLL.” We then took this peptide and did MS/MS on thepeptide under identical conditions as previously used. FIG. 18illustrates the spectrum generated from MS/MS of the endogenously loadedreticulocalbin peptide. Matching spectra, as seen here, are indicatorsthat this peptide sequence is GPRTAALGLL as almost every amino acidcombination will generate a completely different set of fragments, bothin terms of production of fragments and in terms of intensity of thosefragments present. FIG. 18 shows the MS/MS endogenous and synthetic“GPRTAALGLL” peptide under identical collision conditions. As can beseen, the MS/MS graphs are virtually identical.

[0163] In accordance with the present invention, one peptide ligand(i.e. “GPRTAALGLL”) has been identified as being presented by the B*0702class I MHC molecule in cells infected with the HIV MN-1 virus but notin uninfected cells. As one of ordinary skill in the art can appreciatethe novelty and usefulness of the present methodology in directlyidentifying such peptide ligands and the importance such identificationhas for numerous therapeutic (vaccine development, drug targeting) anddiagnostic tools. As such, numerous other peptide ligands have beenuniquely identified in cells infected with HIV MN-1 (as opposed touninfected cells_and these results are summarized in TABLE VII. One ofordinary skill in the art given the present specification would be fullyenabled to identify the “GPRTAALGLL” peptide ligand; as well as otheruniquely presented peptide ligands found in cells infected with amicroorganism of interest and/or tumorigenic cells.

[0164] As stated above, TABLE VII identifies the sequences of peptideligands identified to date as being unique to HIV infected cells. ClassI sHLA B*0702 was harvested for T cells infected and not infected withHIV. Peptide ligands were eluted from B*0702 and comparatively mapped ona mass spectrometer so that ions unique to infected cells were apparent.Ions unique to infected cells (and one ligand unique to uninfectedcells) were subjected to mass spectrometric fragmentation for peptidesequencing. Column 1 indicates the ion selected for sequencing, column 2is the HPLC fraction, column 3 is the peptide sequence, column 4 is thepredicted molecular weight, column 5 is the molecular weight we found,column 6 is the source protein for the epitope sequenced, column 7 iswhere the epitope starts in the sequence of the source protein, column 8is the accession number, and column 9 is a descriptor which brieflyindicates what is known of that epitope and/or its source protein.

[0165] The methodology used herein is to use sHLA to determine what isunique to unhealthy cells as compared to healthy cells. Using sHLA tosurvey the contents of a cell provides a look at what is unique tounhealthy cells in terms of proteins that are processed into peptides.TABLE VII shows the utility of the method described herein fordiscovering epitopes and their source proteins which are unique to HIVinfected cells. A detailed description of the peptide fromReticulocalbin is provided hereinabove. The other epitopes andcorresponding source proteins described in TABLE VII were processed inthe same manner as the reticulocalbin epitope and source proteinwere—i.e. as described herein above. The data summarized in TABLE VIIshows that the epitope discovery technique described herein is capableof identifying sHLA bound epitopes and their corresponding sourceproteins which are unique to infected/unhealthy cells.

[0166] Likewise, and as is shown in TABLE VII, peptide ligands presentedin individual class I MHC molecules in an uninfected cell that are notpresented by individual class I MHC molecules in an uninfected cell canalso be identified. The peptide “GSHSMRY”, for example, was identifiedby the method of the present invention as being an individual class IMHC molecule which is presented in an uninfected cell but not in aninfected cell.

[0167] The utility of this data is at least threefold. First, the dataindicates what comes out of the cell with HLA. Such data can be used totarget CTL to unhealthy cells. Second, antibodies can be targeted tospecifically recognize HLA molecules carrying the ligand described.Third, realization of the source protein can lead to therapies anddiagnostics which target the source protein. Thus, an epitope unique tounhealthy cells also indicates that the source protein is unique in theunhealthy cell.

[0168] The methods of epitope discovery and comparative ligand mappingdescribed herein are not limited to cells infected by a microorganismsuch as HIV. Unhealthy cells analyzed by the epitope discovery processdescribed herein can arise from virus infection or also canceroustransformation. In addition, the status of an unhealthy cell can also bemimicked by transfecting a particular gene known to be expressed duringviral infection or tumor formation. For example, particular genes of HIVcan be expressed in a cell line as described (Achour, A., et al., AIDSRes Hum Retroviruses, 1994. 10(1): p. 19-25; and Chiba, M., et al., CTL.Arch Virol, 1999. 144(8): p. 1469-85, all of which are expresslyincorporated herein by reference) and then the epitope discovery processperformed to identify how the expression of the transferred genemodifies epitope presentation by sHLA. In a similar fashion, genes knownto be upregulated during cancer (Smith, E. S., et al., Nat Med, 2001.7(8): p. 967-72, which is expressly incorporated herein by reference)can be transferred in cells with sHLA and epitope discovery thencompleted. Thus, epitope discovery with sHLA as described herein can becompleted on cells infected with intact pathogens, cancerous cells orcell lines, or cells into which a particular cancer, viral, or bacterialgene has been transferred. In all these instances the sHLA describedhere will provide a means for detecting what changes in terms of epitopepresentation and the source proteins for the epitopes.

[0169] Thus, in accordance with the present invention, there has beenprovided a methodology for epitope discovery and comparative ligandmapping which includes methodology for producing and manipulating ClassI and Class II MHC molecules from gDNA that fully satisfies theobjectives and advantages set forth herein above. Although the inventionhas been described in conjunction with the specific drawings,experimentation, results and language set forth herein above, it isevident that many alternatives, modifications, and variations will beapparent to those skilled in the art. Accordingly, it is intended toembrace all such alternatives, modifications and variations that fallwithin the spirit and broad scope of the invention.

What is claimed is:
 1. A method of isolating peptide ligands for anindividual class I molecule, comprising the steps of: providing a cellline containing a construct that encodes an individual soluble class Imolecule, the cell line being able to naturally process proteins intopeptide ligands capable of being loaded into antigen binding grooves ofclass I molecules; culturing the cell line under conditions which allowfor expression of the individual soluble class I molecules from theconstruct, such conditions also allowing for endogenous loading of apeptide ligand into the antigen binding groove of each individualsoluble class I molecule prior to secretion of the individual solubleclass I molecules from the cell; isolating the secreted individualsoluble class I molecules having the endogenously loaded peptide ligandsbound thereto; and separating the peptide ligands from the individualsoluble class I molecules.
 2. The method of claim 1 wherein, in the stepof providing a cell line containing a construct that encodes anindividual soluble class I molecule, the construct further encodes a tagwhich is attached to the individual soluble class I molecule and aids inisolating the individual soluble class I molecule.
 3. The method ofclaim 2, wherein the tag is selected from the group consisting of a HIStail and a FLAG tail.
 4. The method of claim 1 wherein, in the step ofproviding a cell line, the cell line is class I negative.
 5. The methodof claim 1 wherein, in the step of providing a cell line, the cell lineexpresses endogenous class I molecules.
 6. The method of claim 1wherein, in the step of providing a cell line, the cell line is infectedwith at least one of a microorganism, a gene from a microorganism, or atumor gene.
 7. The method of claim 6 wherein, the cell line is infectedwith HIV.
 8. The method of claim 1 wherein, in the step of providing acell line, the cell line is transformed such that the cell line is atumor cell line.
 9. The method of claim 1 wherein, in the step ofproviding a cell line containing a construct that encodes an individualsoluble class I molecule, the cell line containing the construct thatencodes the individual soluble class I molecule is produced by a methodcomprising the steps of: obtaining genomic DNA or cDNA encoding at leastone class I molecule; identifying an allele encoding an individual classI molecule in the genomic DNA or cDNA; PCR amplifying the alleleencoding the individual class I molecule in a locus specific manner suchthat a PCR product produced therefrom encodes a truncated, soluble formof the individual class I molecule; cloning the PCR product into anexpression vector, thereby forming a construct that encodes theindividual soluble class I molecule; and transfecting the construct intoa cell line.
 10. The method of claim 9 wherein, in the step of providinga cell line containing a construct that encodes an individual solubleclass I molecule, the construct further encodes a tag which is attachedto the soluble class I molecule and aids in isolating the individualsoluble class I molecule.
 11. The method of claim 10, wherein the tag isselected from the group consisting of a HIS tail and a FLAG tail. 12.The method of claim 10, wherein the tag is encoded by a PCR primerutilized in the step of PCR amplifying the allele encoding theindividual class I molecule.
 13. The method of claim 10, wherein the tagis encoded by the expression vector into which the PCR product iscloned.
 14. The method of claim 1, further comprising the step ofidentifying the peptide ligand.
 15. The method of claim 14, furthercomprising the step of identifying a source protein from which thepeptide ligand was obtained.
 16. A peptide ligand for an individualclass I molecule isolated by a method comprising the steps of: providinga cell line containing a construct that encodes an individual solubleclass I molecule, the cell line being able to naturally process proteinsinto peptide ligands capable of being loaded into antigen bindinggrooves of class I molecules; culturing the cell line under conditionswhich allow for expression of the individual soluble class I moleculesfrom the construct, such conditions also allowing for endogenous loadingof a peptide ligand into the antigen binding groove of each individualsoluble class I molecule prior to secretion of the individual solubleclass I molecules from the cell; isolating the secreted individualsoluble class I molecules having the endogenously loaded peptide ligandsbound thereto; and separating the peptide ligands from the individualsoluble class I molecules.
 17. A peptide ligand for an individual classI molecule comprising SEQ ID NO:29.
 18. The peptide ligand of claim 17,wherein the peptide ligand is obtained from HIV-1 MN, ENV.
 19. A peptideligand for an individual class I molecule comprising SEQ ID NO:30. 20.The peptide ligand of claim 19, wherein the peptide ligand is obtainedfrom Cholinergic Receptor, Alpha-3 Polypeptide.
 21. A peptide ligand foran individual class I molecule comprising SEQ ID NO:31.
 22. The peptideligand of claim 21, wherein the peptide ligand is obtained fromUbiquitin-Specific Protease.
 23. A peptide ligand for an individualclass I molecule comprising SEQ ID NO:32.
 24. The peptide ligand ofclaim 23, wherein the peptide ligand is obtained from β-AssociatedTranscript Protein 3 (BAT3).
 25. A peptide ligand for an individualclass I molecule comprising SEQ ID NO:33.
 26. The peptide ligand ofclaim 25, wherein the peptide ligand is obtained from HLA-B Heavy ChainLeader Sequence.
 27. A peptide ligand for an individual class I moleculecomprising SEQ ID NO:34.
 28. A peptide ligand for an individual class Imolecule comprising SEQ ID NO:35.
 29. The peptide ligand of claim 28,wherein the peptide ligand is obtained from RNA Polymerase IIPolypeptide A.
 30. A peptide ligand for an individual class I moleculecomprising SEQ ID NO:36.
 31. The peptide ligand of claim 30, wherein thepeptide ligand is obtained from EUK, Translation Initiation Factor 4.32. A peptide ligand for an individual class I molecule comprising SEQID NO:37.
 33. The peptide ligand of claim 32, wherein the peptide ligandis obtained from SPARC-1 Like Protein.
 34. A peptide ligand for anindividual class I molecule comprising SEQ ID NO:38.
 35. The peptideligand of claim 34, wherein the peptide ligand is obtained fromTenascin-C.
 36. A peptide ligand for an individual class I moleculecomprising SEQ ID NO:39.
 37. The peptide ligand of claim 36, wherein thepeptide ligand is obtained from Polypyrimidine Tract-Binding Protein 1.38. A peptide ligand for an individual class I molecule comprising SEQID NO:40.
 39. The peptide ligand of claim 38, wherein the peptide ligandis obtained from Reticulocalbin.
 40. A peptide ligand for an individualclass I molecule comprising SEQ ID NO:41.
 41. The peptide ligand ofclaim 40, wherein the peptide ligand is obtained from ELAV (HuR).
 42. Amethod for identifying at least one endogenously loaded peptide ligandthat distinguishes an infected cell from an uninfected cell, comprisingthe steps of: providing an uninfected cell line containing a constructthat encodes an individual soluble class I molecule, the uninfected cellline being able to naturally process proteins into peptide ligandscapable of being loaded into antigen binding grooves of class Imolecules; infecting a portion of the uninfected cell line with at leastone of a microorganism, a gene from a microorganism or a tumor gene,thereby providing an infected cell line; culturing the uninfected cellline and the infected cell line under conditions which allow forexpression of individual soluble class I molecules from the construct,such conditions also allowing for endogenous loading of a peptide ligandin the antigen binding groove of each individual soluble class Imolecule prior to secretion of the individual soluble class I moleculesfrom the cell; isolating the secreted individual soluble class Imolecules having the endogenously loaded peptide ligands bound theretofrom the uninfected cell line and the infected cell line; separating theendogenously loaded peptide ligands from the individual soluble class Imolecules from the uninfected cell line and separating the endogenouslyloaded peptide ligands from the individual soluble class I moleculesfrom the infected cell line; isolating the endogenously loaded peptideligands from the uninfected cell line and the endogenously loadedpeptide ligands from the infected cell line; comparing the endogenouslyloaded peptide ligands isolated from the infected cell line to theendogenously loaded peptide ligands isolated from the uninfected cellline; and identifying at least one endogenously loaded peptide ligandpresented by the individual soluble class I molecule on the infectedcell line that is not presented by the individual soluble class Imolecule on the uninfected cell line.
 43. The method of claim 42wherein, in the step of providing an uninfected cell line containing aconstruct that encodes an individual soluble class I molecule, theconstruct further encodes a tag which is attached to the individualsoluble class I molecule and aids in isolating the individual solubleclass I molecule.
 44. The method of claim 42 wherein, the uninfectedcell line is class I negative.
 45. The method of claim 42 wherein, theuninfected cell line expresses endogenous class I molecules.
 46. Themethod of claim 42 wherein, in the step of providing an uninfected cellline containing a construct that encodes an individual soluble class Imolecule, the uninfected cell line containing the construct that encodesthe individual soluble class I molecule is produced by a methodcomprising the steps of: obtaining genomic DNA or cDNA encoding at leastone class I molecule; identifying an allele encoding an individual classI molecule in the genomic DNA or cDNA; PCR amplifying the alleleencoding the individual class I molecule in a locus specific manner suchthat a PCR product produced therefrom encodes a truncated, soluble formof the individual class I molecule; cloning the PCR product into anexpression vector, thereby forming a construct that encodes theindividual soluble class I molecule; and transfecting the construct intoan uninfected cell line.
 47. The method of claim 46 wherein, in the stepof providing an uninfected cell line containing a construct that encodesan individual soluble class I molecule, the construct further encodes atag which is attached to the soluble class I molecule and aids inisolating the individual soluble class I molecule.
 48. The method ofclaim 47, wherein the tag is selected from the group consisting of a HIStail and a FLAG tail.
 49. The method of claim 47, wherein the tag isencoded by a PCR primer utilized in the step of PCR amplifying an alleleencoding the individual class I molecule.
 50. The method of claim 47,wherein the tag is encoded by the expression vector into which the PCRproduct is cloned.
 51. The method of claim 42, further comprising thestep of identifying a source protein from which the at least oneendogenously loaded peptide ligand presented by the individual solubleclass I molecule on the infected cell line and not presented by theindividual soluble class I molecule on the uninfected cell line isobtained.
 52. The method of claim 42 wherein, in the step of identifyingat least one endogenously loaded peptide ligand presented by theindividual soluble class I molecule on the infected cell line but not onthe uninfected cell line, the at least one endogenously loaded peptideligand is obtained from a protein encoded by at least one of themicroorganism, the gene from a microorganism or the tumor gene withwhich the cell line was infected to form the infected cell line.
 53. Themethod of claim 42 wherein, in the step of identifying at least oneendogenously loaded peptide ligand presented by the individual solubleclass I molecule on the infected cell line but not on the uninfectedcell line, the at least one endogenously loaded peptide ligand isobtained from a protein encoded by the uninfected cell line.
 54. Themethod of claim 53, wherein the protein encoded by the uninfected cellline from which the at least one endogenously loaded peptide ligand isobtained has increased expression in a tumor cell line.
 55. The methodof claim 42 wherein, in the step of infecting a portion of theuninfected cell line, the portion of the uninfected cell line isinfected with HIV.
 56. A method for identifying at least oneendogenously loaded peptide ligand that distinguishes an infected cellfrom an uninfected cell, comprising the steps of: providing anuninfected cell line containing a construct that encodes an individualsoluble class I molecule, the uninfected cell line being able tonaturally process proteins into peptide ligands capable of being loadedinto antigen binding grooves of class I molecules; infecting a portionof the uninfected cell line with at least one of a microorganism, a genefrom a microorganism or a tumor gene, thereby providing an infected cellline; culturing the uninfected cell line and the infected cell lineunder conditions which allow for expression of the individual solubleclass I molecules from the construct, such conditions also allowing forendogenous loading of a peptide ligand into the antigen binding grooveof each individual soluble class I molecule prior to secretion of theindividual soluble class I molecules from the cell; isolating thesecreted individual soluble class I molecules having the endogenouslyloaded peptide ligands bound thereto from the uninfected cell line andthe infected cell line; separating the endogenously loaded peptideligands from the individual soluble class I molecules from theuninfected cell line and separating the endogenously loaded peptideligands from the individual soluble class I molecules from the infectedcell line; isolating the endogenously loaded peptide ligands from theuninfected cell line and the endogenously loaded peptide ligands fromthe infected cell line; comparing the endogenously loaded peptideligands isolated from the uninfected cell line to the endogenouslyloaded peptide ligands isolated from the infected cell line; andidentifying at least one endogenously loaded peptide ligand presented bythe individual soluble class I molecule on the uninfected cell line thatis not presented by the individual soluble class I molecule on theinfected cell line.
 57. The method of claim 56 wherein, in the step ofproviding an uninfected cell line containing a construct that encodes anindividual soluble class I molecule, the construct further encodes a tagwhich is attached to the individual soluble class I molecule and aids inisolating the individual soluble class I molecule.
 58. The method ofclaim 56, wherein the uninfected cell line is class I negative.
 59. Themethod of claim 56, wherein the uninfected cell line expressesendogenous class I molecules.
 60. The method of claim 56 wherein, in thestep of providing an uninfected cell line containing a construct thatencodes an individual soluble class I molecule, the uninfected cell linecontaining the construct that encodes the individual soluble class Imolecule is produced by a method comprising the steps of: obtaininggenomic DNA or cDNA encoding at least one class I molecule; identifyingan allele encoding an individual class I molecule in the genomic DNA orcDNA; PCR amplifying the allele encoding the individual class I moleculein a locus specific manner such that a PCR product produced therefromencodes a truncated, soluble form of the individual class I molecule;cloning the PCR product into an expression vector, thereby forming aconstruct that encodes the individual soluble class I molecule; andtransfecting the construct into an uninfected cell line.
 61. The methodof claim 60 wherein, in the step of providing an uninfected cell linecontaining a construct that encodes an individual soluble class Imolecule, the construct further encodes a tag which is attached to theindividual soluble class I molecule and aids in isolating the individualsoluble class I molecule.
 62. The method of claim 61, wherein the tag isselected from the group consisting of a HIS tail and a FLAG tail. 63.The method of claim 61, wherein the tag is encoded by a PCR primerutilized in the step of PCR amplifying the allele encoding theindividual class I molecule.
 64. The method of claim 62 wherein the tagis encoded by the expression vector into which the PCR product iscloned.
 65. The method of claim 56, further comprising the step ofidentifying a source protein from which the at least one endogenouslyloaded peptide ligand presented by the individual soluble class Imolecule on the uninfected cell line and not presented by the individualsoluble class I molecule on the infected cell line is obtained.
 66. Themethod of claim 56 wherein, in the step of infecting a portion of theuninfected cell line, the portion of the uninfected cell line isinfected with HIV.
 67. An endogenously loaded peptide ligand presentedby an individual class I molecule on an infected cell but not on anuninfected cell.
 68. The endogenously loaded peptide ligand of claim 67,wherein the endogenously loaded peptide ligand comprises SEQ ID NO:29.69. The endogenously loaded peptide ligand of claim 67, wherein theendogenously loaded peptide ligand comprises SEQ ID NO:30.
 70. Theendogenously loaded peptide ligand of claim 67, wherein the endogenouslyloaded peptide ligand comprises SEQ ID NO:31.
 71. The endogenouslyloaded peptide ligand of claim 67, wherein the endogenously loadedpeptide ligand comprises SEQ ID NO:32.
 72. The endogenously loadedpeptide ligand of claim 67, wherein the endogenously loaded peptideligand comprises SEQ ID NO:33.
 73. The endogenously loaded peptideligand of claim 67, wherein the endogenously loaded peptide ligandcomprises SEQ ID NO:34.
 74. The endogenously loaded peptide ligand ofclaim 67, wherein the endogenously loaded peptide ligand comprises SEQID NO:35.
 75. The endogenously loaded peptide ligand of claim 67,wherein the endogenously loaded peptide ligand comprises SEQ ID NO:36.76. The endogenously loaded peptide ligand of claim 67, wherein theendogenously loaded peptide ligand comprises SEQ ID NO:37.
 77. Theendogenously loaded peptide ligand of claim 67, wherein the endogenouslyloaded peptide ligand comprises SEQ ID NO:38.
 78. The endogenouslyloaded peptide ligand of claim 67, wherein the endogenously loadedpeptide ligand comprises SEQ ID NO:39.
 79. The endogenously loadedpeptide ligand of claim 67, wherein the endogenously loaded peptideligand comprises SEQ ID NO:40.
 80. The endogenously loaded peptideligand of claim 67, wherein the endogenously loaded peptide ligandcomprises SEQ ID NO:41.
 81. A self protein capable of being naturallyprocessed into at least one peptide fragment, wherein the at least onepeptide fragment is endogenously loaded in and presented by a class Imolecule on an infected cell.
 82. The self protein of claim 81 whereinthe at least one peptide fragment is not endogenously loaded in andpresented by a class I molecule on an uninfected cell.
 83. A peptideligand presented by an individual class I molecule on an infected cellbut not on an uninfected cell, the peptide ligand identified by a methodcomprising the steps of: providing an uninfected cell line containing aconstruct that encodes an individual soluble class I molecule, the cellline being able to naturally process proteins into peptide ligandscapable of being loaded into antigen binding grooves of class Imolecules; infecting a portion of the uninfected cell line with at leastone of a microorganism, a gene from a microorganism or a tumor gene,thereby providing an infected cell line; culturing the uninfected cellline and the infected cell line under conditions which allow forexpression of the individual soluble class I molecules from theconstruct, such conditions also allowing for endogenous loading of apeptide ligand in the antigen binding groove of each individual solubleclass I molecule prior to secretion of the individual soluble class Imolecules from the cell; isolating the secreted individual soluble classI molecules having the endogenously loaded peptide ligands bound theretofrom the uninfected cell line and the infected cell line; separating theendogenously loaded peptide ligands from the individual soluble class Imolecules from the uninfected cell and the endogenously loaded peptideligands from the individual soluble class I molecules from the infectedcell; isolating the endogenously loaded peptide ligands from theuninfected cell line and the endogenously loaded peptide ligands fromthe infected cell line; comparing the endogenously loaded peptideligands isolated from the infected cell line to the endogenously loadedpeptide ligands isolated from the uninfected cell line; and identifyingat least one endogenously loaded peptide ligand presented by theindividual soluble class I molecule on the infected cell line that isnot presented by the individual soluble class I molecule on theuninfected cell line.
 84. The peptide ligand of claim 83 wherein, in thestep of providing an uninfected cell line containing a construct thatencodes an individual soluble class I molecule, the uninfected cell linecontaining the construct that encodes the individual soluble class Imolecule is produced by a method comprising the steps of: obtaininggenomic DNA or cDNA encoding at least one class I molecule; identifyingan allele encoding an individual class I molecule in the genomic DNA orcDNA; PCR amplifying the allele encoding the individual class I moleculein a locus specific manner such that a PCR product produced therefromencodes a truncated, soluble form of the individual class I molecule;cloning the PCR product into an expression vector, thereby forming aconstruct that encodes the individual soluble class I molecule; andtransfecting the construct into an uninfected cell line.
 85. The peptideligand of claim 84, wherein the construct further encodes a tag which isattached to the individual soluble class I molecule and aids inisolating the individual soluble class I molecule.
 86. The peptideligand of claim 85, wherein the tag is selected from the groupconsisting of a HIS tail and a FLAG tail.
 87. The peptide ligand ofclaim 85, wherein the tag is encoded by a PCR primer utilized in thestep of PCR amplifying the allele encoding the individual class Imolecule.
 88. The peptide ligand of claim 85, wherein the tag is encodedby the expression vector into which the PCR product is cloned.
 89. Thepeptide ligand of claim 83, wherein the at least one endogenously loadedpeptide ligand is obtained from a protein encoded by at least one of themicroorganism, the gene from the microorganism or the tumor gene withwhich the portion of the uninfected cell line is infected to form theinfected cell line.
 90. The peptide ligand of claim 83, wherein the atleast one endogenously loaded peptide ligand is obtained from a proteinencoded by the uninfected cell line.
 91. The peptide ligand of claim 83,wherein the portion of the uninfected cell line is infected with HIV.92. A source protein from which the peptide ligand of claim 83 isobtained.
 93. A peptide ligand endogenously loaded in an individualclass I molecule and presented by the individual class I molecule on anuninfected cell.
 94. The peptide ligand of claim 93 wherein the peptideligand is not endogenously loaded in the individual class I molecule andpresented by the individual class I molecule on an infected cell.
 95. Aself protein capable of being processed into at least one peptidefragment, wherein the at least one peptide fragment is endogenouslyloaded in an individual class I molecule and presented by the individualclass I molecule on an uninfected cell.
 96. The self protein of claim 95wherein the at least one peptide fragment is not endogenously loaded inthe individual class I molecule and presented by the individual class Imolecule on an infected cell.
 97. A peptide ligand endogenously loadedin an individual class I molecule and presented by the individual classI molecule on an uninfected cell but not on an infected cell, thepeptide ligand identified by a method comprising the steps of: providingan uninfected cell line containing a construct that encodes anindividual soluble class I molecule, the uninfected cell line being ableto naturally process proteins into peptide ligands capable of beingloaded into antigen binding grooves of class I molecules; infecting aportion of the uninfected cell line with at least one of amicroorganism, a gene from a microorganism or a tumor gene, therebyproviding an infected cell line; culturing the uninfected cell line andthe infected cell line under conditions which allow for expression ofthe individual soluble class I molecules, such conditions also allowingfor endogenous loading of a peptide ligand in the antigen binding grooveof each individual soluble class I molecule prior to secretion of theindividual soluble class I molecules from the cell; isolating thesecreted individual soluble class I molecules having the endogenouslyloaded peptide ligands bound thereto from the uninfected cell line andthe infected cell line; separating the endogenously loaded peptideligands from the individual soluble class I molecules from theuninfected cell line and the infected cell line; isolating theendogenously loaded peptide ligands from the uninfected cell line andthe endogenously loaded peptide ligands from the infected cell line;comparing the endogenously loaded peptide ligands isolated from theuninfected cell line to the endogenously loaded peptide ligands isolatedfrom the infected cell line; and identifying at least one endogenouslyloaded peptide ligand presented by the individual soluble class Imolecule on the uninfected cell line that is not presented by theindividual soluble class I molecule on the infected cell line.
 98. Thepeptide ligand of claim 97 wherein the construct further encodes a tagwhich is attached to the individual soluble class I molecule and aids inisolating the individual soluble class I molecule.
 99. The peptideligand of claim 97 wherein the uninfected cell line containing theconstruct that encodes the individual soluble class I molecule isproduced by a method comprising the steps of: obtaining genomic DNA orcDNA encoding at least one class I molecule; identifying an alleleencoding an individual class I molecule in the genomic DNA or cDNA; PCRamplifying the allele encoding the individual class I molecule in alocus specific manner such that a PCR product produced therefrom encodesa truncated, soluble form of the individual class I molecule; cloningthe PCR product into an expression vector, thereby forming a constructthat encodes an individual soluble class I molecule; and transfectingthe construct into an uninfected cell line.
 100. The peptide ligand ofclaim 99, wherein the construct further encodes a tag which is attachedto the individual soluble class I molecule and aids in isolating theindividual soluble class I molecule.
 101. The peptide ligand of claim99, wherein the tag is selected from the group consisting of a HIS tailand a FLAG tail.
 102. The peptide ligand of claim 99, wherein the tag isencoded by a PCR primer utilized in the step of PCR amplifying an alleleencoding the individual class I molecule.
 103. The method of claim 99,wherein the tag is encoded by the expression vector into which the PCRproduct is cloned.
 104. A source protein from which the peptide ligandof claim 97 is obtained.
 105. A method for identifying a self proteinthat is processed into at least one peptide fragment, wherein the atleast one peptide fragment is endogenously loaded in an individual classI molecule and presented by the individual class I molecule on aninfected cell but not on an uninfected cell, the method comprising thesteps of: providing an uninfected cell line containing a construct thatencodes an individual soluble class I molecule, the uninfected cell linebeing able to naturally process proteins into peptide ligands capable ofbeing loaded into antigen binding grooves of class I molecules;infecting a portion of the uninfected cell line with at least one of amicroorganism, a gene from a microorganism or a tumor gene, therebyproviding an infected cell line; culturing the uninfected cell line andthe infected cell line under conditions which allow for expression ofthe individual soluble class I molecules, such conditions also allowingfor endogenous loading of a peptide ligand in the antigen binding grooveof each individual soluble class I molecule prior to secretion of theindividual soluble class I molecules from the cell; isolating thesecreted individual soluble class I molecules having endogenously loadedpeptide ligands bound thereto from the uninfected cell line and theinfected cell line; separating the endogenously loaded peptide ligandsfrom the individual soluble class I molecules from the uninfected cellline and the infected cell line; isolating the endogenously loadedpeptide ligands from the uninfected cell line and the endogenouslyloaded peptide ligands from the infected cell line; comparing theendogenously loaded peptide ligands isolated from the infected cell lineto the endogenously loaded peptide ligands isolated from the uninfectedcell line; identifying at least one endogenously loaded peptide ligandpresented by the individual soluble class I molecule on the infectedcell line that is not presented by the individual soluble class Imolecule on the uninfected cell line; determining the source proteinfrom which the at least one endogenously loaded peptide ligand isobtained; and identifying the source protein as a self protein if thesource protein is not encoded by the microorganism, gene from amicroorganism or tumor gene with which the infected cell line isinfected but is encoded by the uninfected cell line.
 106. A method foridentifying a self protein that is processed into at least one peptidefragment, wherein the at least one peptide fragment is endogenouslyloaded in an individual class I molecule presented by the individualclass I molecule on an uninfected cell but not on an infected cell, themethod comprising the steps of: providing an uninfected cell linecontaining a construct that encodes an individual soluble class Imolecule, the uninfected cell line being able to naturally processproteins into peptide ligands capable of being loaded into antigenbinding grooves of class I molecules; infecting a portion of theuninfected cell line with at least one of a microorganism, a gene from amicroorganism or a tumor gene, thereby providing an infected cell line;culturing the uninfected cell line and the infected cell line underconditions which allow for expression of the individual soluble class Imolecules, such conditions also allowing for endogenous loading of apeptide ligand in the antigen binding groove of each individual solubleclass I molecule prior to secretion of the individual soluble class Imolecules from the cell; isolating the secreted individual soluble classI molecules having endogenously loaded peptide ligands bound theretofrom the uninfected cell line and from the infected cell line;separating the endogenously loaded peptide ligands from the solubleclass I molecules from the uninfected cell line and from the infectedcell line; isolating the endogenously loaded peptide ligands from theuninfected cell line and the endogenously loaded peptide ligands fromthe infected cell line; comparing the endogenously loaded peptideligands isolated from the uninfected cell line to the endogenouslyloaded peptide ligands isolated from the infected cell line; identifyingat least one endogenously loaded peptide ligand presented by theindividual soluble class I molecule on the uninfected cell line that isnot presented by the individual soluble class I molecule on the infectedcell line; and determining the source protein from which the at leastone endogenously loaded peptide ligand is obtained.
 107. A kit foridentifying endogenously loaded peptide ligands for an individual classI molecule, comprising: a cell line containing a construct that encodesan individual soluble class I molecule, the cell line capable ofnaturally processing proteins into peptide ligands capable of beingloaded into antigen binding grooves of class I molecules such that whenthe cell line is cultured under conditions which allow for expression ofthe individual soluble class I molecule, a peptide ligand isendogenously loaded into the antigen binding groove of each individualsoluble class I molecule prior to secretion of the individual solubleclass I molecules from the cell.
 108. The kit of claim 107 wherein thecell line is produced by a method comprising the steps of: obtaininggenomic DNA or cDNA encoding at least one class I molecule; identifyingan allele encoding an individual class I molecule in the genomic DNA orcDNA; PCR amplifying the allele encoding the individual class I moleculein a locus specific manner such that a PCR product produced therefromencodes a truncated, soluble form of the individual class I molecule;cloning the PCR product into an expression vector, thereby forming aconstruct that encodes an individual soluble class I molecule; andtransfecting the construct into an uninfected cell line.
 109. A methodfor detecting a disease state, comprising the step of: providing meansfor detecting an endogenously loaded peptide ligand in an individualclass I molecule, wherein the endogenously loaded peptide ligand ispresented by an individual class I molecule on an infected cell but noton an uninfected cell.
 110. The method of claim 109, wherein theendogenously loaded peptide ligand comprises SEQ ID NO:29.
 111. Themethod of claim 109, wherein the endogenously loaded peptide ligandcomprises SEQ ID NO:30.
 112. The method of claim 109, wherein theendogenously loaded peptide ligand comprises SEQ ID NO:31.
 113. Themethod of claim 109, wherein the endogenously loaded peptide ligandcomprises SEQ ID NO:32.
 114. The method of claim 109, wherein theendogenously loaded peptide ligand comprises SEQ ID NO:33.
 115. Themethod of claim 109, wherein the endogenously loaded peptide ligandcomprises SEQ ID NO:34.
 116. The method of claim 109, wherein theendogenously loaded peptide ligand comprises SEQ ID NO:35.
 117. Themethod of claim 109, wherein the endogenously loaded peptide ligandcomprises SEQ ID NO:36.
 118. The method of claim 109, wherein theendogenously loaded peptide ligand comprises SEQ ID NO:37.
 119. Themethod of claim 109, wherein the endogenously loaded peptide ligandcomprises SEQ ID NO:38.
 120. The method of claim 109, wherein theendogenously loaded peptide ligand comprises SEQ ID NO:39.
 121. Themethod of claim 109, wherein the endogenously loaded peptide ligandcomprises SEQ ID NO:40.
 122. The method of claim 109, wherein theendogenously loaded peptide ligand comprises SEQ ID NO:41.
 123. Themethod of claim 109, wherein the disease state is HIV infection.
 124. Amethod for detecting a disease state, comprising the step of: providingmeans for detecting an endogenously loaded peptide ligand in anindividual class I molecule, wherein the endogenously loaded peptideligand is presented by the individual class I molecule on an uninfectedcell but not on an infected cell.
 125. A kit for detecting a diseasestate, comprising: means for detecting an endogenously loaded peptideligand in an individual class I molecule, wherein the endogenouslyloaded peptide ligand is presented by the individual class I molecule onan infected cell but not on an uninfected cell.
 126. The method of claim125, wherein the endogenously loaded peptide ligand comprises SEQ IDNO:29.
 127. The method of claim 125, wherein the endogenously loadedpeptide ligand comprises SEQ ID NO:30.
 128. The method of claim 125,wherein the endogenously loaded peptide ligand comprises SEQ ID NO:31.129. The method of claim 125, wherein the endogenously loaded peptideligand comprises SEQ ID NO:32.
 130. The method of claim 125, wherein theendogenously loaded peptide ligand comprises SEQ ID NO:33.
 131. Themethod of claim 125, wherein the endogenously loaded peptide ligandcomprises SEQ ID NO:34.
 132. The method of claim 125, wherein theendogenously loaded peptide ligand comprises SEQ ID NO:35.
 133. Themethod of claim 125, wherein the endogenously loaded peptide ligandcomprises SEQ ID NO:36.
 135. The method of claim 125, wherein theendogenously loaded peptide ligand comprises SEQ ID NO:37.
 136. Themethod of claim 125, wherein the endogenously loaded peptide ligandcomprises SEQ ID NO:38.
 137. The method of claim 125, wherein theendogenously loaded peptide ligand comprises SEQ ID NO:39.
 138. Themethod of claim 125, wherein the endogenously loaded peptide ligandcomprises SEQ ID NO:40.
 139. The method of claim 125, wherein theendogenously loaded peptide ligand comprises SEQ ID NO:41.
 140. A kitfor detecting a disease state, comprising: means for detecting a peptideligand endogenously loaded in an individual class I molecule, whereinthe peptide ligand is presented by the individual class I molecule on anuninfected cell but not on an infected cell.
 141. A method ofidentifying a motif for endogenously loaded peptide ligands presented byan individual class I molecule, comprising the steps of: providing acell line containing a construct that encodes an individual solubleclass I molecule, the cell line being able to naturally process proteinsinto peptide ligands capable of being loaded into antigen bindinggrooves of class I molecules; culturing the cell line under conditionswhich allow for expression of the individual soluble class I moleculefrom the construct, such conditions also allowing for endogenous loadingof the peptide ligand into the antigen binding groove of each individualsoluble class I molecule prior to secretion of the individual solubleclass I molecules from the cell; isolating the secreted individualsoluble class I molecules having endogenously loaded peptide ligandsbound thereto; separating the endogenously loaded peptide ligands fromthe individual soluble class I molecules, thereby forming a pool ofendogenously loaded peptide ligands; and sequencing the pool ofendogenously loaded peptide ligands and deriving a motif for theendogenously loaded peptide ligands based on dominant occurrences ofparticular amino acids at specific positions of the endogenously loadedpeptide ligands.
 142. A method of targeting a compound to an infectedcell, comprising the steps of: providing an uninfected cell linecontaining a construct that encodes an individual soluble class Imolecule, the cell line being able to naturally process proteins intopeptide ligands capable of being loaded into antigen binding grooves ofclass I molecules; infecting a portion of such cell line with at leastone of a microorganism, a gene from a microorganism or a tumor gene,thereby providing an infected cell line; culturing the uninfected cellline and the infected cell line under conditions which allow forexpression of the individual soluble class I molecule, such conditionsalso allowing for endogenous loading of a peptide ligand in the antigenbinding groove of each individual soluble class I molecule prior tosecretion of the individual soluble class I molecules from the cell;isolating the secreted individual soluble class I molecules havingendogenously loaded peptide ligands bound thereto from the uninfectedcell line and the infected cell line; separating the endogenously loadedpeptide ligands from the individual soluble class I molecules from theuninfected cell line and the infected cell line; isolating theendogenously loaded peptide ligands from the uninfected cell line andthe endogenously loaded peptide ligands from the infected cell line;comparing the endogenously loaded peptide ligands isolated from theinfected cell line to the endogenously loaded peptide ligands isolatedfrom the uninfected cell line; and identifying at least one endogenouslyloaded peptide ligand presented by the individual soluble class Imolecule on the infected cell line that is not presented by theindividual soluble class I molecule on the uninfected cell line; andtargeting a compound to a cell having the at least one endogenouslyloaded peptide ligand presented by the individual class I molecule on asurface of the cell.
 143. The method of claim 142 wherein, in the stepof targeting a compound to a cell, the compound is a drug.
 144. Themethod of claim 142 wherein, in the step of targeting a compound to acell, the compound is an antibody.
 145. The method of claim 142 wherein,in the step of targeting a compound to a cell, the compound specificallyrecognizes a complex formed of the at least one endogenously loadedpeptide ligand and the individual class I molecule.